Modified FRT Recombination Site Libraries and Methods of Use

ABSTRACT

Methods and compositions using populations of randomized modified FRT recombination sites to identify, isolate and/or characterize modified FRT recombination sites are provided. Kits comprising the library populations of FRT sites are also provided, as are methods to make a library of modified FRT recombination sites. The recombinogenic modified FRT recombination sites can be employed in a variety of methods for targeted recombination of polynucleotides of interest.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is an continuing application of U.S. application Ser.No. 11/487,273 filed Jul. 14, 2006, and claims the benefit of U.S.Application Ser. No. 60/700,225 filed Jul. 18, 2005, the disclosure ofeach is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to site-specific recombination systems and methodsof use.

BACKGROUND

The random insertion of introduced DNA into the genome of a host cellcan be lethal if the foreign DNA happens to insert into, and therebymutate, a critically important native gene. In addition, even if arandom insertion event does not impair the functioning of a gene of ahost cell, the expression of an inserted foreign nucleotide sequence maybe influenced by position effects caused by the surrounding genomic DNA.In some cases, the nucleotide sequence is inserted into a site where theposition effect is strong enough to suppress the function or regulationof the introduced nucleotide sequence. In other instances,overproduction of the gene product has deleterious effects on a cell.

For example, in plants, position effects can result in reducedagronomics, additional costs for further research, creation ofadditional transgenic events, and slower time to product. For thesereasons, efficient methods are needed for targeting the insertion ofnucleotide sequences into the genome of various organisms, such asplants, at chromosomal positions that allow desired function of thesequence of interest.

SUMMARY

Methods and compositions using populations of randomized modified FRTrecombination sites to identify, isolate and/or characterize modifiedFRT recombination sites are provided. Kits comprising the librarypopulations of FRT sites are also provided, as are methods to make alibrary of modified FRT recombination sites. The recombinogenic modifiedFRT recombination sites can be employed in a variety of methods fortargeted recombination of polynucleotides of interest.

DETAILED DESCRIPTION

Methods and compositions using modified FRT recombination sites include,but are not limited to the following:

1. A method to select a recombinogenic modified FRT recombination sitecomprising:a) providing a first population of plasmids wherein each plasmid in saidfirst population comprises a common first selectable marker; and, eachplasmid in said first population comprises a member of a population ofmodified FRT recombination sites;b) providing a second population of plasmids wherein each plasmid insaid second population comprises a common second selectable marker,wherein said first and said second selectable markers are distinct; and,each plasmid in said second population comprises a member of thepopulation of modified FRT recombination sites;c) combining said first population of plasmids with said secondpopulation of plasmids in the presence of a FLP recombinase underconditions where site-specific recombination can occur; and,d) selecting for a co-integrant plasmid comprising the first and thesecond selectable marker, said co-integrate plasmid comprising themodified FRT recombination site.2. The method of 1, wherein each member of said population of randomizedmodified FRT recombination sites comprises a spacer region comprising atleast one nucleotide alteration in SEQ ID NO:43.3. The method of 1, wherein said first and said second population ofplasmids are combined in the presence of the FLP recombinase.4. The method of 1, wherein said co-integrant plasmid comprises afunctional modified FRT recombination site.5. The method of 1 further comprising isolating the co-integrantplasmid.6. The method of 1, further comprising characterizing the modified FRTrecombination site of said co-integrant plasmid.7. The method of 6, wherein characterizing the modified FRTrecombination site comprises determining excision efficiency.8. The method of 6, wherein characterizing the modified FRTrecombination site comprises determining recombination specificity.9. The method of 6, wherein characterizing the modified FRTrecombination sites comprises sequencing the modified FRT recombinationsite of the co-integrant plasmid.10. The method of 1, wherein at least one of said first or said secondselectable markers is selected from the group consisting of ampicillinand spectinomycin.11. The method of 1, wherein said first population of plasmids and saidsecond population of plasmids are combined in an equimolar ratio.12. An isolated library comprising a population of plasmids wherein eachplasmid in said population comprises a common selectable marker; and,each plasmid in said population comprises a member of a population ofmodified FRT recombination sites, wherein said population of plasmidscomprises at least about 5 distinct members of the plasmid population.13. The isolated library of 12, wherein each member of said populationof modified FRT recombination sites comprises a spacer region comprisingat least one nucleotide alteration in SEQ ID NO:43.14. The isolated library of 12 or 13, wherein said modified FRTrecombination sites of said population are recombinogenic.15. The isolated library of any one of 12-14, wherein the selectablemarker is selected from the group consisting of ampicillin andspectinomycin.16. The isolated library of any one of 12-15, wherein said population ofplasmids comprises at least about 100 distinct members of the plasmidpopulation.17. The isolated library of any one of 12-15, wherein said population ofplasmids comprises at least one modified recombinogenic FRT sitecomprising a spacer region selected from the group consisting of SEQ IDNOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18.18. The isolated library of 17, wherein said population of plasmidscomprises at least one modified recombinogenic FRT site selected fromthe group consisting of SEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, or 38.19. A kit comprisinga) a first population of plasmids wherein each plasmid in said firstpopulation comprises a first common selectable marker; and, each plasmidin said first population comprises a member of a population of modifiedFRT recombination sites; and,b) a second population of plasmids wherein each plasmid in said secondpopulation comprises a second common selectable marker, wherein saidfirst and said second selectable markers are distinct; and, each plasmidin said second population comprises a member of the population ofmodified FRT recombination sites.20. The kit of 19, wherein said kit further comprises a FLP recombinaseor a polynucleotide encoding said FLP recombinase.21. The kit of 20, wherein said kit comprises the polynucleotideencoding a biologically active variant of the FLP recombinase or abiologically active fragment of the FLP recombinase.22. The kit of any one of 19-21, wherein at least one member of saidfirst population, or said second population of modified FRTrecombination sites, or both populations comprisesa) a spacer region comprising at least one nucleotide alteration in SEQID NO:43; andb) a spacer region selected from the group consisting of SEQ ID NOS:1,2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 21, 22, 23,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.23. The kit of any one of 19-22, wherein each member of said first andsaid second population of modified FRT recombination site isrecombinogenic.24. The kit of any one of 19-22, wherein at least one of said first orsaid second selectable marker is selected from the group consisting ofampicillin and spectinomycin.25. A method for generating a library of molecules comprisinga) providing a population of modified FRT recombination sites; and,b) contacting said population of modified FRT recombination sites with apopulation of plasmids having a common selectable marker underconditions for the insertion of said population of modified FRTrecombination sites into said population of plasmids, such that each ofthe plasmids of said population comprises a single member of thepopulation of modified FRT recombination sites, whereby a library ofmolecules is generated.26. The method of 25, wherein each member of said population of modifiedFRT recombination sites comprises a spacer region comprising at leastone nucleotide alteration in SEQ ID NO:43.27. An isolated polynucleotide comprising a nucleotide sequencecomprising at least one functional modified FRT recombination site, saidfunctional modified FRT recombination site comprising a spacer sequenceselected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, and 18.28. The isolated polynucleotide of 27, wherein said functional modifiedFRT recombination site comprises SEQ ID NO:21, 22, 23, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or a functional variantthereof, wherein said functional variant is substantially identical toSEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, or 38.29. The isolated polynucleotide of 28, wherein said functional modifiedFRT recombination site comprises SEQ ID NO:21, 22, 23, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.30. The isolated polynucleotide of 27, 28, or 29, wherein saidpolynucleotide comprises a second recombination site.31. The isolated polynucleotide of 30, wherein said second recombinationsite is selected from the group consisting of a FRT site, a mutant FRTsite, a LOX site, or a mutant LOX site.32. The isolated polynucleotide of 31, wherein said second recombinationsite is selected from the group consisting SEQ ID NO:1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.33. The isolated polynucleotide of any one of 30-32, wherein said secondrecombination site is dissimilar and non-recombinogenic with respect tothe functional modified FRT recombination site.34. The isolated polynucleotide of any one of 30-32, wherein saidfunctional modified FRT recombination site and said second recombinationsite are corresponding recombination sites.35. A cell comprising the polynucleotide of any one of 27-34.36. The cell of 35, wherein said cell is from a plant.37. The cell of 35 or 36, wherein the polynucleotide is stablyintegrated into the genome of said cell.38. The cell of 36, wherein said cell is from a monocotyledonous plant.39. The cell of 38, wherein said monocotyledonous plant cell is frommaize, barley, millet, wheat, sorghum, rye, or rice.40. The cell of 36, wherein said plant cell is from a dicotyledonousplant.41. The cell of 40, wherein said dicotyledonous plant cell is fromsoybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, orcotton.42. A plant comprising the cell of any one of 36-41.43. A seed having stably integrated into its genome the polynucleotideof any one of 27-34.44. The cell of any one of 35-42, wherein said cell further has stablyincorporated into its genome a polynucleotide encoding a FLPrecombinase.45. The cell of 44, wherein said polynucleotide encodes a biologicallyactive variant of the FLP recombinase.46. A method for determining the relative recombination excisionefficiency of a first and a second FRT recombination site comprisinga) providing a polynucleotide comprising the first and the second FRTrecombination site, wherein the spacer sequence of said first or saidsecond FRT recombination site is selected from the group consisting ofSEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and18;b) providing a FLP recombinase under conditions such that said FLPrecombinase implements a recombinase-mediated excision event; and,c) determining excision efficiency of said first and said second FRTrecombination site relative to a control reaction, wherein the controlreaction is done under identical conditions using wild type FRTrecombination sites as the first and the second FRT recombination sites.47. The method of 46, wherein said first and said second FRTrecombination sites are corresponding recombination sites.48. A method to identify dissimilar and non-recombinogenic recombinationsites comprisinga) providing a first FRT recombination site wherein the spacer sequenceis selected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, and 18;b) providing a second dissimilar FRT recombination site;c) providing a FLP recombinase under conditions such that said FLPrecombinase implements a recombination event; and,d) assaying for a recombination event to thereby determine if the firstand the second recombination site are non-recombinogenic.49. The method of any one of 46-48, wherein said first FRT recombinationsite comprises SEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38 or a functional variant thereof, wherein saidfunctional variant is substantially identical to SEQ ID NO:21, 22, 23,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.50. The method of 49, wherein said first FRT recombination sitecomprises SEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, or 38.51. The method of 46-48, wherein said method occurs in vivo.52. The method of 51, wherein providing one or more of said first FRTsite or said second FRT site comprises transformation.53. A method for producing site-specific recombination of DNA comprisinga) providing a first DNA fragment comprising a first site-specificrecombination site, wherein the first site-specific recombination sitecomprises a polynucleotide is selected from the group consisting of:i) a FRT recombination site having a spacer region selected from thegroup consisting of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, and 18; andii) a FRT recombination site selected from the group consisting of SEQID NOS: 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,and 38;b) providing a second DNA fragment comprising a second site-specificrecombination site which is recombinogenic with the first site-specificrecombination site; and,c) providing a site-specific recombinase that catalyzes a site-specificrecombination between the first and the second site-specificrecombination sites.54. The method of 53 wherein the first DNA fragment and the second DNAfragment are provided on a single polynucleotide molecule.55. The method of 53 or 54 wherein the first site-specific recombinationsite and the second site-specific recombination sites are correspondingsites.56. The method of 53 or 54 wherein the first site-specific recombinationsite and the second site-specific recombination sites are dissimilarsites.57. The method of any one of 53-56 wherein the first site-specificrecombination site and the second site-specific recombination site aredirectly oriented relative to each other.58. The method of 57 wherein the first and the second site-specificrecombination sites flank a first polynucleotide of interest, wherebyproviding the site-specific recombinase excises the first polynucleotideof interest.59. The method of 58 wherein excision of the first polynucleotide ofinterest activates expression of a second polynucleotide of interest.60. The method of any one of 54-56 wherein the first site-specificrecombination site and the second site-specific recombination site arein the opposite orientation relative to each other.61. The method of 60 wherein the first and the second site-specificrecombination sites flank a first polynucleotide of interest, wherebyproviding the site-specific recombinase inverts the first polynucleotideof interest.62. The method of 61 wherein inversion of the first polynucleotide ofinterest activates expression of the first polynucleotide of interest.63. The method of 61 wherein inversion of the first polynucleotide ofinterest activates expression of a second polynucleotide of interest.64. The method of 53 wherein the first DNA fragment is provided on afirst polynucleotide and the second DNA fragment is provided on a secondseparate polynucleotide.65. The method of 64, wherein the second polynucleotide is a circularmolecule.66. The method of 64 or 65 wherein the second polynucleotide furthercomprises a polynucleotide of interest.67. The method of any one of 53-66 wherein the second site-specificrecombination site is a modified FRT recombination site comprising apolynucleotide selected from the group consisting of SEQ ID NOS:1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38.68. The method of any one of 53-67 wherein the site-specificrecombination occurs in vivo.69. The method of 68 wherein the site-specific recombination occurs in aeukaryotic cell.70. The method of 69 wherein the eukaryotic cell is a plant cell.71. The method of 70 wherein the plant cell is from a plant selectedfrom the group consisting of maize, rice, wheat, barley, millet,sorghum, rye, soybean, alfalfa, canola, Arabidopsis, tobacco, sunflower,cotton, and safflower.72. A method for targeting the insertion of a polynucleotide of interestto a target site comprisinga) providing the target site comprising a first functional recombinationsite comprising a spacer sequence selected from the group consisting ofSEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and18;b) providing a transfer cassette comprising a second functionalrecombination site and said polynucleotide of interest, wherein saidfirst and said second recombination sites are recombinogenic withrespect to one another; and,c) providing a recombinase wherein said recombinase recognizes andimplements recombination at the first and the second recombinationsites, and the polynucleotide of interest is inserted at the targetsite.73. A method for targeting the insertion of a polynucleotide of interestto a target site, said method comprising:a) providing the target site comprising a first and a second functionalrecombination site, wherein said first and said second recombinationsites are dissimilar and non-recombinogenic with respect to one another;and at least one of said first or said second recombination sitescomprises a spacer sequence selected from the group consisting of SEQ IDNOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18;b) providing a transfer cassette comprising the polynucleotide ofinterest, wherein said polynucleotide of interest is flanked by saidfirst and said second recombination sites, and,c) providing a recombinase, wherein said recombinase recognizes andimplements recombination at the first and the second recombinationsites, and the polynucleotide of interest is inserted at the targetsite.74. The method of 73, wherein said target site comprises a secondpolynucleotide of interest flanked by said first and said secondrecombination site.75. A method for assessing promoter activity in a cell comprising:a) providing the cell having in its genome a target site comprising afirst and a second functional recombination site, wherein said first andsaid second recombination sites are dissimilar and non-recombinogenic,and at least one of said first or said second recombination sitescomprises a spacer sequence selected from the group consisting of SEQ IDNOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18; and,b) providing to said cell a transfer cassette comprising a promoteroperably linked to a polynucleotide comprising a selectable marker,wherein said transfer cassette is flanked by the first and the secondrecombination sites,c) providing a recombinase, wherein said recombinase recognizes andimplements recombination at the first and the second recombinationsites, whereby said transfer cassette is integrated at the target site;and,d) monitoring expression of the selectable marker to assess promoteractivity.76. A method to directly select a transformed cell, said methodcomprising:a) providing a population of cells comprising a polynucleotidecomprising, in the following order, a promoter operably linked to atarget site, wherein the target site comprises a first recombinationsite and a second recombination site, said first and said secondrecombination sites are dissimilar and non-recombinogenic with respectto one another, and at least one of said first or said secondrecombination sites comprises a spacer sequence selected from the groupconsisting of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, and 18;b) introducing into said population of cells a transfer cassettecomprising in the following order the first recombination site, apolynucleotide encoding a selectable marker gene not operably linked toa promoter, and the second recombination site;c) providing a recombinase, wherein said recombinase recognizes andimplements recombination at the first and the second recombinationsites; and,d) growing said population of cells on an appropriate selective agent todirectly select the cell expressing the selectable marker.77. The method of any one of 72-76, wherein said target site is stablyincorporated into the genome of a cell.78. A method to minimize or eliminate expression resulting from randomintegration of a nucleic acid molecule of interest into a genome of acell comprising:a) providing the cell having stably incorporated into its genome apolynucleotide comprising in the following order: a promoter active insaid cell operably linked to an ATG translational start site operablylinked to a target site comprising a first and a second functionalrecombination site, wherein said first and said second recombinationsites are dissimilar and non-recombinogenic, and at least one of saidfirst or said second recombination sites comprise a spacer sequenceselected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, and 18;b) providing to said cell a transfer cassette comprising in thefollowing order: the first recombination site, the nucleic acid moleculeof interest, and the second recombination site, wherein the ATGtranslational start site of the nucleic acid molecule of interest hasbeen replaced with said first recombination site; and,c) providing a recombinase, wherein said recombinase recognizes andimplements recombination at the first and the second recombinationsites, whereby the nucleic acid molecule of interest is integrated atthe target site and thereby operably linked to the promoter andtranslational start site of the polynucleotide.79. A method to excise or invert a polynucleotide of interest in a cellcomprising:a) providing a cell having a transfer cassette comprising thepolynucleotide of interest flanked by a first and a second functionalrecombination site, wherein said first and said second recombinationsites are dissimilar and non-recombinogenic and wherein and at least oneof said first or said second recombination sites comprise a spacersequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18;b) providing to said cell an isolated oligonucleotide capable ofdirecting a nucleotide conversion in one of the first or the secondrecombination sites so as to create two corresponding recombinationsites; and,c) providing a recombinase, wherein said recombinase that implementsrecombination at the corresponding recombination sites, whereby thepolynucleotide of interest is excised or inverted.80. The method of 79, wherein said corresponding recombination sites aredirectly repeated.81. The method of 79, wherein said corresponding recombination sites areinverted.82. The method of 79, wherein said polynucleotide of interest is apromoter or encodes a polypeptide.83. A method for locating preferred integration sites within the genomeof a cell, said method comprisinga) introducing into said cell a target site comprising in the followingorder: a first functional recombination site, a promoter active in saidcell operably linked to a polynucleotide, and a second functionalrecombination site, wherein said first and said second recombinationsites are dissimilar and non-recombinogenic and wherein and at least oneof said first or said second recombination sites comprise a spacersequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18;b) determining the level of expression of said polynucleotide; and,c) selecting the cell expressing said polynucleotide.84. The method of 83, further comprising introducing into the cell atransfer cassette comprising a polynucleotide of interest flanked bysaid first and said second recombination sites; and, providing arecombinase, wherein said recombinase recognizes and implementsrecombination at the first and the second recombination sites, wherebythe transfer cassette is integrated at the preferred site.85. The method of any one of 75-84 wherein said cell has stablyincorporated into its genome a polynucleotide encoding said recombinase.86. The method of any one of 73-84 wherein at least one of saiddissimilar and non-recombinogenic recombination sites is selected fromthe group consisting of a FRT site, a functional variant of the FRTsite, a LOX site, and a functional variant of the LOX site.87. The method of 86, wherein one of said dissimilar andnon-recombinogenic recombination sites comprises a FRT site or afunctional variant of the FRT site.88. The method of 87, wherein said functional variant of the FRT site isselected from the group consisting of SEQ ID NO:21, 22, 23, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, or 42.89. The method of any one of 73-84 wherein said recombinase is a FLPrecombinase or a Cre recombinase.90. The method of 89, wherein the FLP recombinase or the Cre recombinaseis encoded by a polynucleotide having maize preferred codons.91. The method of 89, wherein said recombinase comprises a FLPrecombinase or a Cre recombinase.92. The method of any one of 73, 74, 75, 77 or 78, wherein providingsaid transfer cassette comprises transformation.93. The method of any one of 73, 74, 75, 77 or 78, wherein providingsaid transfer cassette comprises sexual breeding.94. The method of any one of 72-79 or 84, wherein providing saidrecombinase comprises transformation.95. The method of any one of 72-79 or 84, wherein providing saidrecombinase comprises sexual breeding.96. The method of any one of 72-78, 83, or 84, wherein at least one ofsaid first or said second recombination sites comprises SEQ ID NO:21,22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or afunctional variant thereof, wherein said functional variant issubstantially identical to SEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, or 38.97. The method of 96, wherein at least one of said first or said secondrecombination site comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NOS:21, 22, 23, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, and 38.98. A method to combine multiple transfer cassettes comprising:a) providing a target site comprising at least a first and a secondfunctional recombination site;b) providing a first transfer cassette comprising in the following orderat least the first, a third, and the second functional recombinationsites, wherein the first and the third recombination sites of the firsttransfer cassette flank a first polynucleotide of interest, said first,said second, and said third recombination sites are dissimilar andnon-recombinogenic with respect to one another, and at least one of saidfirst, said second, or said third recombination sites comprises a spacersequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18;c) providing a first recombinase, wherein said first recombinaserecognizes and implements recombination at the first and the secondrecombination sites;d) providing a second transfer cassette comprising at least the secondand the third recombination sites, wherein the second and the thirdrecombination sites of the second transfer cassette flank a secondpolynucleotide of interest; and,e) providing a second recombinase, wherein said second recombinaserecognizes and implements recombination at the second and thirdrecombination sites, whereby the first and the second transfer cassettesare integrated at the target site.99. A method to combine multiple transfer cassettes comprising:a) providing a target site comprising in the following order at least afirst, a second, and a third functional recombination site; wherein saidfirst, said second, and said third recombination sites are dissimilarand non-recombinogenic with respect to one another, and at least one ofsaid first, said second, or said third recombination sites comprise aspacer sequence selected from the group consisting of SEQ ID NOS:1, 2,3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18;b) providing a first transfer cassette comprising a first polynucleotideof interest flanked by the first and the second recombination sites;c) providing a first recombinase, wherein said first recombinase orvariant thereof recognizes and implements recombination at the first andthe second recombination sites;d) providing a second transfer cassette comprising a secondpolynucleotide of interest flanked by at least the second and the thirdrecombination sites; and,e) providing a second recombinase, wherein said second recombinaserecognizes and implements recombination at the second and thirdrecombination sites, whereby the first and the second transfer cassettesare integrated at the target site.100. The method of 98 or 99, wherein said target site is in a cell.101. The method of 100, wherein said target site is stably incorporatedinto the genome of the cell.102. The method of 98 or 99, wherein at least one of said first, saidsecond, or said first and said second recombinase comprises a FLPrecombinase.103. The method of 102, wherein said first or said second recombinasefurther comprises a Cre recombinase.104. The method of 100, wherein at least one polynucleotide encoding atleast said first or said second recombinase is stably incorporated intothe genome of the cell.105. The method of 97 or 98, wherein at least one of said dissimilar andnon-recombinogenic recombination sites is selected from the groupconsisting of a FRT site, a biologically active variant of the FRT site,a LOX site, and a biologically active variant of the LOX site.106. The method of 105, wherein one of said dissimilar andnon-recombinogenic recombination sites comprises a FRT site or abiologically active variant of the FRT site.107. The method of 106, wherein said biologically active variant of theFRT site is FRT 5 (SEQ ID NO:40). FRT 6 (SEQ ID NO:41), FRT 7 (SEQ IDNO:42), or FRT 87 (SEQ ID NO: 24).108. The method of 102 or 103, wherein the FLP recombinase or the Crerecombinase is encoded by a polynucleotide having maize preferredcodons.109. The method of 102, wherein said first, said second, or said firstand said second recombinase comprises a FLP recombinase.110. The method of 102, wherein said first, said second, or said firstand said second recombinase comprises a Cre recombinase.111. The method 98 or 99, wherein providing at least one of said firstor said second recombinase comprises transformation.112. The method of 98 or 99, wherein providing at least one of saidfirst or said second recombinase comprises sexual breeding.113. The method 98 or 99, wherein introducing at least one of said firstor said second transfer cassette comprises transformation.114. The method of 98 or 99, wherein introducing at least one of saidfirst or said second transfer cassette comprises sexual breeding.115. The method of 98 or 99, wherein at least one of said first, saidsecond, or said third recombination sites comprises SEQ ID NO:21, 22,23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or afunctional variant thereof, wherein said functional variant issubstantially identical to SEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, or 38.116. The method of 115, wherein at least one of said first, said second,or said third recombination site comprises SEQ ID NO:21, 22, 23, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.117. A method for inverting a polynucleotide of interest comprisinga) providing a target site comprising the polynucleotide of interestflanked by a first and a second recombination site, said first and saidsecond recombination sites are recombinogenic with respect to oneanother and are in an inverted orientation relative to each other; andat least one of said first and said second recombination site comprisesa spacer sequence selected from the group consisting of SEQ ID NOS:1, 2,3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18; and,b) providing a FLP recombinase, wherein said FLP recombinase recognizesand implements recombination at the first and the second recombinationsites, thereby inverting the polynucleotide of interest.118. A method to excise a polynucleotide of interest comprisinga) providing a target site comprising a polynucleotide of interestflanked by a first and a second recombination site, said first and saidsecond recombination sites are recombinogenic with respect to oneanother and are in a directly repeated orientation relative to eachother; and at least one of said first and said second recombination sitecomprises a spacer sequence selected from the group consisting of SEQ IDNOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18;b) providing a FLP recombinase, wherein said FLP recombinase recognizesand implements recombination at the first and the second recombinationsites, thereby excising the polynucleotide of interest.119. The method of 117 or 118, wherein said target site is in a cell.120. The method of 119, wherein said target site is stably incorporatedinto the genome of the cell.121. The method of any one of 75, 76, 77, 79, 82, 90, 91, 119 or 120,wherein said cell is a plant cell.122. The method of 119, wherein said method occurs in a cell havingstably incorporated into its genome a nucleotide sequence encoding saidFLP recombinase.123. The method of any one of 113, 117, 118, or 122 wherein the FLPrecombinase is encoded by a polynucleotide having maize preferredcodons.124. The method of any one of 113, 117 or 118, wherein said recombinasecomprises the FLP recombinase.125. The method of 119, wherein providing said target site comprisestransformation.126. The method of 119, wherein providing said target site comprisessexual breeding.127. The method 119, wherein providing said FLP recombinase comprisestransformation.128. The method of 119, wherein providing said FLP recombinase comprisessexual breeding.129. The method of any one of 113, 117 or 118, wherein said first orsaid second recombination sites comprise SEQ ID NO:21, 22, 23, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or a functionalvariant thereof, wherein said functional variant is substantiallyidentical to SEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, or 38.130. The method of 129, wherein said first or said second recombinationsites comprise a nucleotide sequence selected from the group consistingof SEQ ID NOS:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, and 38.131. The method of 121, wherein said plant cell is a monocotyledonousplant cell.132. The method of 131, wherein said monocotyledonous cell is frommaize, barley, millet, wheat, sorghum, rye, or rice.133. The method of 121, wherein said plant cell is a dicotyledonousplant cell.134. The method of 133, wherein said dicotyledonous cell is fromsoybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, orcotton.135. The method of any one of 72, 117, or 118, wherein said first andsaid second recombination sites are corresponding.136. The method of 72 or 135, wherein said first and said secondrecombination sites comprise SEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, or 38 or a functional variant thereof,wherein said functional variant is substantially identical to SEQ IDNO:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or38.137. The method of 136, wherein said first and said second recombinationsites comprise a nucleotide sequence selected from the group consistingof SEQ ID NOS:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, and 38.138. The method of 73, wherein said target site is operably linked to afirst and a second convergent promoter; said transfer cassettecomprises, in the following order, the first functional recombinationsite, a polynucleotide of interest orientated in the 5′ to 3′ direction,a second polynucleotide of interest oriented in the 3′ to 5′ direction,and the second functional recombination site; wherein insertion of thetransfer cassette at the target site results in the first polynucleotideof interest operably linked to the first convergent promoter and thesecond polynucleotide of interest operably linked to the secondconvergent promoter.139. The isolated polynucleotide of 30, wherein said polynucleotidecomprises a first convergent promoter, the first recombination site, thesecond recombination site, and the second convergent promoter.140. The isolated polynucleotide of 139, wherein said polynucleotidecomprises the first convergent promoter, the first recombination site, afirst polynucleotide sequence of interest operably linked to said firstconvergent promoter, a second polynucleotide of interest operably linkedto said convergent promoter and the second convergent promoter.141. A method of excising or inverting a polynucleotide of interestcomprisinga) providing a polynucleotide comprising, in the following order, afirst functional recombination site comprising a spacer sequenceselected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17 and 18, the polynucleotide of interest,and a second functional recombination site, wherein said first and saidsecond recombination sites are recombinogenic with respect to oneanother; and,b) providing a recombinase or a biologically active variant of saidrecombinase, wherein said recombinase recognizes and implementsrecombination at the first and the second recombination sites, whereinthe polynucleotide sequence of interest is excised or inverted.142. The method of 141, wherein said first and said second recombinationsites are identical.143. The method of 141 or 142, wherein said first recombination site,said second recombination site, or both comprise SEQ ID NO:21, 22, 23,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or afunctional variant thereof, wherein said functional variant issubstantially identical to SEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, or 38.144. The method of 143, wherein said first recombination site, saidsecond recombination site, or both comprise a nucleotide sequenceselected from the group consisting of SEQ ID NOS:21, 22, 23, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38.145. The method of any one of 1-11, 46, 48, 52-82, 84, 85, 89, 94, 95,98-138, or 141-144, wherein said recombinase is a biologically activevariant of the native recombinase.

Various populations of modified FRT recombination sites are provided,including, for example, an isolated library of molecules comprising apopulation of plasmids where each plasmid in the population comprises acommon selectable marker; and, each plasmid in the population comprisesa member of a population of modified FRT recombination sites. Thepopulation can comprise at least about 5 distinct members of the plasmidpopulation. Other compositions include, an isolated library where themembers of the population of modified FRT recombination sites comprise avariant of a spacer region as set forth in SEQ ID NO:43, wherein thevariant comprises at least one nucleotide alteration in SEQ ID NO:43.Other compositions include an isolated library where the modified FRTrecombination sites comprise a population of functional modified FRTrecombination sites.

Compositions further include kits comprising two populations ofplasmids. The plasmids in the first population comprise a first commonselectable marker; and, each of the plasmids in the first populationcomprises a member of a population of modified FRT recombination sites.The second population of plasmids comprises a common second and distinctselectable marker; and, each of the plasmids in the second populationcomprises a member of the population of modified FRT recombinationsites. The kit can further comprise a FLP recombinase, a biologicallyactive variant of the FLP recombinase, a polynucleotide encoding a FLPrecombinase, or a polynucleotide encoding a biologically active variantof the FLP recombinase.

Methods to select a recombinogenic modified FRT recombination site arealso provided. The method comprises providing a first population ofplasmids where each of the plasmids in the first population comprises acommon first selectable marker; and, each of the plasmids in the firstpopulation comprises a member of a population of modified FRTrecombination sites. A second population of plasmids is provided whereeach of the plasmids in the second population comprises a common seconddistinct selectable marker; and, each of the plasmids in the secondpopulation comprises a member of the population of modified FRTrecombination sites. The first population of plasmids is combined withthe second population of plasmids in the presence of a FLP recombinaseor a biologically active variant of the FLP recombinase, underconditions that allow for recombinase-mediated integration. Aco-integrant plasmid comprising both the first and the second selectablemarker is selected, wherein the co-integrant plasmid comprises at leastone modified FRT recombination site.

Methods further comprise isolating the co-integrant plasmid and/orcharacterizing the modified FRT recombination site of the co-integrantplasmid. Characterizing the modified FRT recombination site can comprisedetermining recombination excision efficiency and/or determining thesequence of the modified FRT recombination site.

A method for generating a library of molecules is further provided. Themethod comprises providing a population of modified FRT recombinationsites; and, contacting the population of modified FRT recombinationsites with a population of plasmids having a common selectable markerunder conditions that allow for the insertion of the population ofmodified FRT recombination sites into the population of plasmids, suchthat each of the plasmids of the population comprises a member of thepopulation of modified FRT recombination sites.

Additional compositions include an isolated polynucleotide comprising atleast one functional modified FRT recombination site, where thefunctional modified FRT recombination site comprises a spacer sequenceselected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, and 18. Other compositions include anisolated polynucleotide comprising a nucleotide sequence comprising atleast one functional modified FRT recombination site comprising thenucleotide sequence set forth in SEQ ID NOS:21, 22, 23, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or a functional variantthereof, where the variant has substantial sequence identity to SEQ IDNOS:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or38.

Organisms, including, for example, prokaryotes, such as bacteria, andeukaryotes, such as yeast, mammals, insects, worms, plants, plant cells,and seed comprising the recited polynucleotides comprising a modifiedFRT recombination site are also provided. In specific examples, thepolynucleotides are stably integrated into the genome of the organism.

Methods are also provided, including methods for producing site-specificrecombination of DNA. In some examples the site-specific recombinationis an intramolecular reaction, in other examples the site-specificrecombination is an intermolecular reaction. The site-specificrecombination can be done in vitro or in vivo. The in vivo site-specificrecombination reaction can be done in any cell, including prokaryotic oreukaryotic cells. In some examples, the cells are from a plant.Additional methods employ various recombination methods to allow for thetargeted insertion, exchange, alteration, expression, excision and/orinversion of any polynucleotide(s) of interest. In one example, a methodfor targeting the insertion of a polynucleotide of interest to a targetsite is provided. The method comprises providing the target site,wherein the target site comprises a first and a second functionalrecombination site, the first and the second functional recombinationsites are dissimilar and non-recombinogenic with respect to one another;and at least one of the first or the second recombination sitescomprises a modified FRT recombination site disclosed herein. A transfercassette is provided, wherein the transfer cassette comprises thepolynucleotide of interest flanked by the first and the secondrecombination sites. At least one recombinase is provided. Therecombinase recognizes and implements recombination at the first andsecond recombination sites. The method can occur in vitro or in vivo. Inspecific examples, the target site is stably incorporated into thegenome of an organism.

In another example, a method for targeting the insertion of apolynucleotide of interest is provided. The method comprises providing atarget site having at least a first functional recombination site. Atransfer cassette is provided comprising a polynucleotide of interestand at least a second functional recombination site, wherein the secondfunctional recombination site is recombinogenic with the firstfunctional recombination site, and the first and/or the secondrecombination site comprise a modified FRT site disclosed herein. Insome examples, the first and the second recombination sites have thesame sequence. At least one recombinase is provided. The recombinaserecognizes and implements recombination at the first and secondrecombination sites. The method can occur in vitro or in vivo. Inspecific examples, the first functional recombination site is stablyincorporated into the genome of an organism. In some examples, thepolynucleotide of interest and/or the target can later be excised,inverted, or otherwise modified, for example, by the addition of asecond polynucleotide of interest at the target site.

In other examples, methods for assessing promoter activity, methods todirectly select transformed organisms, methods to minimize or eliminateexpression resulting from random integration into the genome of anorganism, such as a plant, methods to excise or invert a polynucleotideof interest, methods to combine multiple transfer cassettes, methods fordetermining the excision efficiency or co-integration efficiency of aset of FRT recombination sites, methods to identify recombinogenic ornon-recombinogenic recombination sites, methods for locating preferredintegration sites within the genome of an organism, methods to recombineDNA molecules both in vitro and in vivo, methods to reduce non-specificagronomic impact of the insertion of a polynucleotide of interest suchas reducing yield drag, and, methods to identify cis regulatory elementsin an organism, such as a plant, are also provided.

The minimal wild type FRT recombination site has been characterized andcomprises a series of domains including the following nucleotidesequence 5′-AGTTCCTATTCTCTAGAAAGTATAGGAACT-3′ (SEQ ID NO:39). Thedomains of the minimal FRT recombination site comprises a pair of 11base pair symmetry elements which are the FLP binding sites (nucleotides1-11 and 20-30 of SEQ ID NO:39); the 8 base pair core, or spacer, region(nucleotides 12-19 of SEQ ID NO:39); and, the polypyrimidine tracts(nucleotides 3-14 and nucleotides 16-29 of SEQ ID NO:39). A modified FRTrecombination site can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morealterations which include substitutions, additions, and/or deletions inone or more of these domains.

Modified FRT recombination sites are provided. A modified FRTrecombination site is a nucleotide sequence that is similar but notidentical to the minimal native FRT recombination site set forth in SEQID NO:39. While the modified FRT recombination site can be functional, amodified FRT recombination site need not retain activity. Unlessotherwise noted, a modified FRT recombination site retains thebiological activity of the wild type FRT recombination site andcomprises a functional recombination site that is recognized by a FLPrecombinase and capable of a recombinase-mediate recombination reaction.Thus, a modified FRT recombination site can comprise a deletion,addition, and/or substitution of one or more nucleotides in the 5′ or 3′end of the minimal native FRT recombination site, in one or moreinternal sites in the minimal native FRT recombination site. Generally,modified recombination sites will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to the minimal native recombination siteover its complete length or to any domain contained therein. Forexample, a modified FRT recombination site will have the recited %sequence identity to the minimal native FRT nucleotide sequence; to thesymmetry elements of the minimal native FRT sequence; to the spacersequence of the wild type FRT sequence; and/or, to the polypyrimidinetract(s) of the minimal native FRT site as determined by sequencealignment programs and parameters described elsewhere herein. Themodified FRT recombination site could therefore include 1, 2, 3, 4, 5,8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29 orgreater nucleotide substitutions, additions, and/or deletions across theentire length of the minimal recombination site, or alternatively, ineach of the various domains of the recombination site as outlined above.

A fragment is a portion of a nucleotide sequence, or of anycharacterized domain contained therein. For example, a fragment of amodified FRT recombination site could be a portion of the minimal nativeFRT recombination site, a portion of one or both of the symmetryelements, a portion of the spacer region and/or a portion of thepolypyrimidine tract(s) of the native FRT site. While the fragments of amodified recombination site need not have biological activity, in someexamples, the fragments of the recombination sites can retain thebiological activity of the recombination site, and hence, the fragmentscan be functional. Unless otherwise noted, a fragment of modified FRTrecombination site retains the biological activity of the wild type FRTrecombination site. For example, fragments of a modified FRTrecombination site may range from at least about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleotides. Fragments of a modified symmetry element sitemay range from at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11nucleotides, fragments of a spacer sequence may range from at leastabout 1, 2, 3, 5, 6, or 7 of a minimal wild type FRT spacer region, andfragments of a polypyrimidine tract can range from at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides.

In other examples, modified FRT recombination sites have mutations suchas alterations, additions, deletions in the 8 base pair spacer domain.Non-limiting examples of modified spacer domains are set forth in SEQ IDNO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. Inspecific examples, the modified FRT sites are functional. In otherexamples, modified FRT recombination sites comprise the spacer regionsset forth in SEQ ID NOS:1-18 and further comprise symmetry element FLPbinding sites that correspond to those found in the minimal native FRTrecombination site. See, SEQ ID NOS:19 and 20 showing wild type symmetryelement sequences. Such modified FRT recombination sites are set forthin SEQ ID NOS:21-38. In specific examples, the modified FRT sites arefunctional. In other examples, modified FRT recombination sites cancomprise the spacer sequence set forth in SEQ ID NOS:1-18 and furthercomprise one or more modifications to the symmetry elements set forth inSEQ ID NOS:19 and 20. In specific examples, the modified FRT sites arefunctional. Modifications of the symmetry elements (nucleotide sequencesat position 1 to 11 and 20 to 30 of SEQ ID NOS:21-38) can include 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more substitutions, additions, deletions, ormodifications of the nucleotide sequence of the wild type symmetryelements set forth in SEQ ID NOS:19 and 20. In other examples, themodifications of the symmetry elements are substantially identical toSEQ ID NOS:21-28. Substantially identical or substantially similarsequence identity refers to a nucleotide sequence having at least one,two, or three substitutions, deletions, and/or additions as compared toa reference sequence. Thus, a substantially identical variant of amodified FRT recombination site is intended a variant of a functionalmodified FRT recombination site comprising the nucleotide sequence setforth in SEQ ID NO:21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,43, 35, 36, 37, or 38, wherein the functional variant comprises A) one,two or three alterations, substitutions, additions, and/or deletionsbetween nucleotide positions 1 to 11 of SEQ ID NO:21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 43, 35, 36, 37, or 38; B) one, two orthree alterations, substitutions, additions, and/or deletions betweennucleotide positions 20 to 30 of SEQ ID NO:21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 43, 35, 36, 37, or 38; and/or C) any combinationof A and B. In specific examples, the modified FRT recombination sitecomprises the spacer sequences of SEQ ID NOS:1-18 and functionalvariants of the symmetry elements. Functional variants of FRT symmetryelements are known, see, for example, Senecoff et al. (1988) J Mol Biol201:406-421 and Voziyanov et al. (2002) Nucleic Acid Res 30:7. Incertain examples, more than one recombination site may be used in acomposition or method.

As discussed above, a modified recombination site can be functional. Afunctional recombination site is a recombination site that isrecombinogenic with a recombination site in the presence of theappropriate recombinase, and unless otherwise noted, a recombinationsite is functional and includes wild type sites, modified sites,variants, and fragments. Methods to determine if a modifiedrecombination site is recombinogenic are known. As used herein, afunctional variant recombination site comprises a functional, modifiedrecombination site.

The recombination sites employed in the methods can be correspondingsites or dissimilar sites. Corresponding recombination sites, or a setof corresponding recombination sites refers to recombination sites havethe same nucleotide sequence. In other examples, the recombination sitesare dissimilar. Dissimilar recombination sites, or a set of dissimilarrecombination sites, are recombination sites that are distinct from eachother by having at least one nucleotide difference. The recombinationsites within a set of dissimilar recombination sites can be eitherrecombinogenic or non-recombinogenic with respect to one another.Recombinogenic refers to recombination sites capable of recombining withone another. Unless otherwise stated, recombinogenic recombination sitesor a set of recombinogenic recombination sites include those sites wherethe relative excision efficiency of recombination between the sites isgreater than 2%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, or greater. Asdefined herein, the relative recombination excision efficiency is theexcision efficiency in the presence of the native recombinase of a firstmodified recombination site with a second modified recombination sitedivided by the excision efficiency of a pair of the appropriate nativerecombination sites×100%. For example, when working with modified FRTsites, the relative recombination excision efficiency is defined as theexcision efficiency in the presence of native FLP (SEQ ID NO:49) of afirst modified FRT site with a second modified FRT site divided by theexcision efficiency of a pair of native FRT sites (FRT1, SEQ ID NO:39).Non-recombinogenic refers to recombination sites which in the presenceof the appropriate recombinase will not recombine with one another, orrecombination between the sites is minimal. Unless otherwise stated,non-recombinogenic recombination sites, or a set of recombinogenicrecombination sites include those sites where the relative excisionefficiency of recombination between the sites is lower than 2%, 1.5%,1%, 0.75%, 0.5%, 0.25%, 0.1%, 0.075, 0.005%, 0.001%. Accordingly, anysuitable set of non-recombinogenic and/or recombinogenic recombinationsites may be utilized, including a FRT site or functional variantthereof, a LOX site or functional variant thereof, any combinationthereof, or any other combination of non-recombinogenic and/orrecombination sites known.

Methods to identify dissimilar and non-recombinogenic recombinationsites are provided. In one method, a first FRT recombination sitecomprising a spacer sequence selected from the group consisting of SEQID NOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 isprovided. A second dissimilar FRT recombination site is provided, alongwith a FLP recombinase under conditions that allow said FLP recombinaseto implement a recombination event. Recombination is assayed todetermine if the first and the second recombination site arenon-recombinogenic with respect to one another. In specific examples,the first and the second recombination sites are provided on the samepolynucleotide, while in other examples, the first and the secondrecombination sites are provided on distinct polynucleotides.

In one example, a method for determining the recombination efficiency,such as relative excision efficiency or the relative co-integrationefficiency of a first and a second FRT recombination sites are provided.For example, a method for determining excision efficiency comprisesproviding a polynucleotide comprising the first and the second FRTrecombination site wherein the spacer sequence for at least the firstand/or the second FRT site is selected from the group consisting of SEQID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18or a variant thereof; and, providing a FLP recombinase under conditionsthat allow the FLP recombinase to implement a recombinase mediatedexcision event. Recombination excision efficiency is determined. Methodsto assay for recombination excision efficiency are known. For example,in Example 3 excision vectors comprising two copies of a modified FRTrecombination site in direct orientation are used. In vivo or in vitroassays can be used to determine if the two modified FRT recombinationsites are capable of mediating excision in the presence of FLPrecombinase.

In another example, a method for determining relative co-integrationefficiency is provided. The method comprises providing a firstpolynucleotide comprising a first FRT recombination site and providing asecond polynucleotide comprising a second FRT recombination site,wherein the spacer sequence of either one or both of the first or secondrecombination site is selected from the group consisting of SEQ IDNOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 ora variant thereof; and, providing a FLP recombinase under conditionsthat allow the FLP recombinase to implement a recombinase mediatedintegration event. Relative co-integration efficiency is determined. Therelative co-integration efficiency of a set of FRT sites is defined asthe co-integration efficiency of the first modified FRT site with asecond FRT site compared to the co-integration efficiency of any givenFRT site chosen as an appropriate standard such as the wild type minimalFRT1 (SEQ ID NO: 39). A functional modified FRT recombination site canhave a co-integration efficiency of about 2%, 10%, 20%, 25%, 30%, 40%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, or greater to the relativestandard, shown for example in Example 4.

In one example, the first and the second FRT recombination sites havecorresponding nucleotide sequences. In yet another example, the firstand the second FRT recombination sites are dissimilar. Therefore varioussets and combinations of FRT recombination sites can be identified suchas sets of functional, dissimilar, non-recombinogenic FRT sites,functional, dissimilar, recombinogenic sites, and/or sets of functional,corresponding, recombinogenic FRT sites.

One or more of the modified FRT recombination sites can be contained ina polynucleotide. In one example, the polynucleotide comprises one ormore expression units. An expression unit is a nucleotide sequencecomprising a unit of DNA characterized by having a singletranscriptional promoter. Alternatively, the polynucleotide containingthe modified FRT recombination site need not contain a promoter and/ordownstream regulatory sequences. In other examples, the polynucleotidecomprising the modified recombination site can be designed such thatupon integration into the genome, the sequences contained in thepolynucleotide are operably linked to an active promoter. It isrecognized that a polynucleotide can have additional elements including,but not limited to, nucleotide sequences of interest, marker genes,recombination sites, termination regions, etc. As illustrated below, thepolynucleotide may comprise transfer cassettes, target sites, or anyportions thereof.

An isolated or purified polynucleotide or protein, or biologicallyactive portion thereof, is substantially or essentially free fromcomponents that normally accompany or interact with the polynucleotideor protein as found in its naturally occurring environment. An isolatedor purified polynucleotide or protein is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Typically, an isolatedpolynucleotide is free of sequences that naturally flank the 5′ and/or3′ ends polynucleotide in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various examples, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% dry weight of contaminating protein. Whenthe protein or biologically active portion thereof is recombinantlyproduced, generally the culture medium represents less than about 30%,20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Polynucleotides can comprise ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides andribonucleotides include both naturally occurring molecules and syntheticanalogues. Polynucleotides also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

In one example, an isolated polynucleotide is provided, wherein thepolynucleotide comprises a modified FRT recombination site. In specificexamples, the modified FRT recombination site is the polynucleotidesequence. For example, an isolated polynucleotide can comprise at leastone functional modified FRT recombination site, where the functionalmodified FRT recombination site comprises a spacer sequence selectedfrom the group consisting of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, and 18 is provided. In specific examples,the modified FRT recombination site is heterologous to thepolynucleotide.

Heterologous refers to a polypeptide or a nucleotide sequence thatoriginates from a different species, or if from the same species, issubstantially modified from its native form in composition and/orgenomic locus. For example, a heterologous recombination site is apolynucleotide is not found in the native polynucleotide or is not foundin the same location in the native polynucleotide, and/or is modifiedfrom its native composition.

In other examples, an isolated polynucleotide is provided comprising anucleotide sequence set forth in SEQ ID NO:21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or a functional variantthereof. In specific examples, the functional variant comprises at leastone, two, three, four, five, six or more alterations between nucleotidepositions 1 to 11 and/or between nucleotide positions 20 to 30 of SEQ IDNO:21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37, or38. In other examples, the functional variant is substantially identicalto the sequence set forth in SEQ ID NO:21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

A modified FRT recombination site can be introduced into an organism ofinterest. Introducing comprises presenting to the organism at least onemolecule, composition, polynucleotide, or polypeptide, in such a mannerthat the composition gains access to the interior of a cell. The methodsdo not depend on a particular method for introducing a polynucleotide orpolypeptide to an organism, only that the polynucleotide or polypeptidegains access to the interior of at least one cell of the organism.

Organisms of interest include, but are not limited to both prokaryoticand eukaryotic organisms including, for example, bacteria, yeast,insects, mammals including mice, humans, and plants. In one example, theorganism is a plant.

Methods for providing or introducing a composition into variousorganisms are known and include but are not limited to, stabletransformation methods, transient transformation methods, virus-mediatedmethods, and sexual breeding. Stable transformation indicates that theintroduced polynucleotide integrates into the genome of the organism andis capable of being inherited by progeny thereof. Transienttransformation indicates that the introduced composition is onlytemporarily expressed or present in the organism.

Protocols for introducing polynucleotides and polypeptides into plantsmay vary depending on the type of plant or plant cell targeted fortransformation, such as monocot or dicot. Suitable methods ofintroducing polynucleotides and polypeptides into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334; and U.S. Pat. No.6,300,543), meristem transformation (U.S. Pat. No. 5,736,369),electroporation (Riggs et al. (1986) Proc Natl Acad Sci USA83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. Nos.5,563,055; and 5,981,840), direct gene transfer (Paszkowski et al.(1984) EMBO J 3:2717-2722), and ballistic particle acceleration (U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al.(1995) “Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe etal. (1988) Biotechnology 6:923-926; Weissinger et al. (1988) Ann RevGenet 22:421-477; Sanford et al. (1987) Particulate Science andTechnology 5:27-37 (onion); Christou et al. (1988) Plant Physiol87:671-674 (soybean); Finer & McMullen (1991) In Vitro Cell Dev Biol27P:175-182 (soybean); Singh et al. (1998) Theor Appl Genet 96:319-324(soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein etal. (1988) Proc Natl Acad Sci USA 85:4305-4309 (maize); Klein et al.(1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855;5,322,783, and 5,324,646; Klein et al. (1988) Plant Physiol 91:440-444(maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize);Hooykaas-Van Slogteren et al. (1984) Nature 311:763-764; U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc Natl Acad Sci USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Rep 9:415-418) andKaeppler et al. (1992) Theor Appl Genet 84:560-566 (whisker-mediatedtransformation); D'Halluin et al (1992) Plant Cell 4:1495-1505(electroporation); Li et al (1993) Plant Cell Rep 12:250-255; Christou &Ford (1995) Annals of Botany 75:407-413 (rice); and, Osjoda et al.(1996) Nat Biotechnol 14:745-750 (maize via Agrobacterium tumefaciens).

Alternatively, the polynucleotides may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating a polynucleotide within a viral DNA or RNAmolecule. It is recognized that a polypeptide of interest may beinitially synthesized as part of a viral polyprotein, which later may beprocessed by proteolysis in vivo or in vitro to produce the desiredrecombinant protein. Further, it is recognized that promoters alsoencompass promoters utilized for transcription by viral RNA polymerases.Methods for introducing polynucleotides into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, areknown, see, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866.785,5,589,367 and 5,316,931.

Transient transformation methods include, but are not limited to, theintroduction of polypeptides such as recombinase protein, directly intothe organism, the introduction of polynucleotides such as DNA and/or RNApolynucleotides, and the introduction of the RNA transcript, such as anmRNA encoding a recombinase, into the organism. Such methods include,for example, microinjection or particle bombardment. See, for example,Crossway et al. (1986) Mol Gen Genet 202:179-185; Nomura et al. (1986)Plant Sci 44:53-58; Hepler et al. (1994) Proc Natl Acad Sci USA91:2176-2180; and, Hush et al. (1994) J Cell Sci 107:775-784.

The cells having the introduced sequence may be grown into plants inaccordance with conventional ways, see, for example, McCormick et al.(1986) Plant Cell Rep 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or with a differentstrain, and the resulting progeny expressing the desired phenotypiccharacteristic and/or comprising the introduced polynucleotide orpolypeptide identified. Two or more generations may be grown to ensurethat the polynucleotide is stably maintained and inherited, and seedsharvested. In this manner, transformed seed, also referred to astransgenic seed, having a polynucleotide, for example, comprising amodified FRT site, stably incorporated into their genome are provided.

Examples of plant genuses and species of interest include, but are notlimited to, monocots and dicots such as corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arechis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats (Avena), barley(Hordeum), palm, legumes including beans and peas such as guar, locustbean, fenugreek, garden beans, cowpea, mungbean, lima bean, fava bean,lentils, chickpea, and castor, Arabidopsis, vegetables, ornamentals,grasses, conifers, crop and grain plants that provide seeds of interest,oil-seed plants, and other leguminous plants. Vegetables includetomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca saliva),green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas(Lathyrus spp.), and members of the genus Cucumis such as cucumber (C.sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophyllahydrangea), hibiscus (Hibiscus rosasenensis), roses (Rosa spp.), tulips(Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),camation (Dianthus caryophyllus), poinsettia (Euphorbia pulchenima), andchrysanthemum. Conifers include, for example, pines such as loblollypine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinusponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinusradiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsugacanadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true first such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

The term plant includes plant cells, plant protoplasts, plant celltissue cultures from which plants can be regenerated, plant calli, plantdumps, and plant cells that are intact in plants or parts of plants suchas embryos, pollen, ovules, seeds, flowers, kernels, ears, cobs, husks,stalks, roots, root tips, anthers, and the like.

Prokaryotic cells may also be used in the methods. Prokaryotes includevarious strains of E. coli; however, other microbial strains may also beused, including, for example, Bacillus sp, Salmonella, andAgrobacterium. Exemplary Agrobacterium strains include C58c1 (pGUSINT),Agt121 (pBUSINT), EHA101 (pMTCA23GUSINT), EHA105 (pMT1), LBA4404(pTOK233), GU2260, BU3600, AGL-1, and LBA4402. Such strains aredescribed in detail in Chan et al. (1992) Plant Cell Physiol 33:577;Smith et al. (1995) Crop Sci 35:301; and Hiei et al. (1994) Plant J6:271-282. Exemplary bacterial strains include, but are not limited to,C600 (ATCC 23724), C600hfi, DH1 (ATCC 33849), DH5α, DH5αF′, ER1727,GM31, GM119 (ATCC 53339), GM2163, HB101 (ATCC 33694), JM83 (ATCC 35607),JM101 (ATCC 33876), JM103 (ATCC 39403), JM105 (ATCC 47016), JM107 (ATCC47014), JM108, JM109 (ATCC53323), JM110 (ATCC 47013), LE392 (ATCC33572), K802 (ATCC 33526), NM522 (ATCC 47000), RR1 (ATCC31343), X1997(ATCC 31244), and Y1088 (ATCC 37195). See also, Jendrisak et al. (1987)Guide to Molecular Cloning Techniques, Academic Press, 359-371, Hanahanet al. (1983) J Mol Biol 166:557-580, Schatz et al. (1989) Cell 59:1035,Bullock et al. (1987) BioTechniques 5:376-378, ATCC Bacteria andBacteriophages (1996) 9 Edition, and Palmer et al. (1994) Gene 143:7-8.

Exemplary, but non-limiting, viral strains include, but are not limitedto, geminivirus, begomovirus, curtovirus, mastrevirus, (−)strand RNAviruses, (+) strand RNA viruses, potyvirus, potexvirus, tobamovirus, orother DNA viruses, nanoviruses, viroids, and the like, for example,African cassava mosaic virus (ACMV) (Ward et al. (1988) EMBO J 7:899-904and Hayes et al. (1988) Nature 334:179-182), barley stripe mosaic virus(BSM) (Joshi et al. (1990) EMBO J 9:2663-2669), cauliflower mosaic virus(CaMV) (Gronenbom et al. (1981) Nature 294:773-776 and Brisson et al.(1984) Nature 310:511-514), maize streak virus (MSV) (Lazarowitz et al.(1989) EMBO J 8:1023-1032 and Shen et al. (1994) J Gen Virol76:965-969), tobacco mosaic virus (TMV) (Takamatsu et al. (1987) EMBO J6:307-311 and Dawson et al. (1989) Virology 172:285-292), tomato goldenmosaic virus (TGMV) (Elmer et al. (1990) Nucleic Acids Res18:2001-2006), and wheat dwarf virus (WDV) (Woolston et al. (1989)Nucleic Acids Res 17:6029-6041) and derivatives thereof. See also, Poratet al. (1996) Mol Biotechnol 5:209-221.

Commonly used prokaryotic control sequences include promoters fortranscription initiation, optionally with an operator, along withribosome binding sequences, include such commonly used promoters as thebeta lactamase (penicillinase) and lactose (lac) promoter systems (Changet al. (1977) Nature 198:1056), the tryptophan (trp) promoter system(Goeddel et al. (1980) Nucleic Acids Res 8:4057) and the lambda derivedP L promoter and N-gene ribosome binding site (Shimatake et al. (1981)Nature 292:128).

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Prokaryotic/bacterial expression systems for expressing a protein areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235; Mosbach et al. (1983) Nature 302:543-545). The Tet operonand the Lac operon can also be employed.

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known for the expression of apolynucleotide of interest. In some examples, transformed/transfectedplant cells are employed as expression systems. Synthesis(introduction/expression) of heterologous nucleotide sequences in yeastis well known (Sherman et al. (1982) Methods in Yeast Genetics, ColdSpring Harbor Laboratory). Two widely utilized yeasts for production ofeukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known and available from commercial suppliers (e.g.,InVitrogen). Suitable vectors usually have expression control sequences,such as promoters, including 3-phosphoglycerate kinase or alcoholoxidase, and an origin of replication, termination sequences and thelike as desired.

Recombinant baculoviruses are generated by inserting the particularsequences-of-interest into the baculovirus genome using establishedprotocols with vectors and reagents from commercial suppliers (e.g.,InVitrogen, Life Technologies Incorporated). Commercial vectors arereadily available with various promoters, such as polyhedrin and p10,optional signal sequences for protein secretion, or affinity tags, suchas 6× histidine. These recombinant viruses are grown, maintained andpropagated in commercially available cell lines derived from severalinsect species including Spodoptera frugiperda and Trichoplusia ni. Theinsect cells can be cultured using well-established protocols in avariety of different media, for example, with and without bovine serumsupplementation. The cultured cells are infected with the recombinantviruses and the sequence-of-interest is expressed. Proteins expressedwith the baculovirus system have been extensively characterized and, inmany cases, their post-translational modifications such asphosphorylation, acylation, etc., are identical to the nativelyexpressed protein.

Compositions further comprise populations of modified FRT recombinationsites. A population is a group or collection that comprises two or more(i.e., 5, 10, 100, 300, 500, 700, 900, 1100, 1300, 1500, 1700, 1900,2100, 2300, 2500, 2700, 2900, 3100, 3300, 3500, 3700, 3900, 4000, 4096,10⁴, 10⁵, 10⁶, 10⁷, 10 ⁸, 10⁹, or greater) dissimilar modified FRTrecombination sites. In specific examples, the modified FRTrecombination sites are heterologous to the polynucleotide. Variouspopulations of modified FRT recombination sites are provided, including,for example, a library of randomized modified FRT recombination sites.The library of modified FRT recombination sites can be used viaselection techniques for the identification of populations offunctional, recombinogenic and/or non-recombinogenic modified FRTrecombination sites.

In one example, the population of modified FRT recombination sitescomprises a library. A library of modified FRT recombination sitescomprises a population of plasmids wherein each of the plasmids in thepopulation comprises a common selectable marker. In addition, each ofthe plasmids in the population comprises a member of the population ofrandomized modified FRT recombination sites. Accordingly, each plasmidin the library population has the potential to contain a dissimilarmember of the randomized modified FRT recombination site. Populations ofmany different modified FRT recombination sites can be screened toidentify recombinogenic modified FRT recombination sites.

Methods of producing or forming a population of randomized modified FRTrecombination sites include identifying the region of the FRTrecombination site in which alterations are desired, such as the entirelength of the FRT site, the symmetry region, the spacer region, thepolypyrimidine tract, or any combination thereof, and, for example,generating a population of oligonucleotides that have the randomlymodified nucleotides at the desired region. The randomized sequences inthe library of modified FRT recombination sites can be of variouslengths and comprise various domains. The chemical or enzymaticreactions by which random sequence segments are made may not yieldmathematically random sequences due to unknown biases or nucleotidepreferences that may exist. The term randomized, or random, reflects thepossibility of such deviations from non-ideality. Accordingly, the termrandomized is used to describe a segment of a nucleic acid having, inprinciple, any possible sequence of nucleotides containing natural ormodified bases over a given length. In addition, a bias can bedeliberately introduced into the randomized sequence, for example, byaltering the molar ratios of precursor nucleoside or deoxynucleosidetriphosphates of the synthesis reaction. A deliberate bias may bedesired, for example, to approximate the proportions of individual basesin a given organism, or to affect secondary structure. See, Hermes etal. (1998) Gene 84:143-151 and Bartel et al. (1991) Cell 67:529-536. Seealso, Davis et al. (2002) Proc Natl Acad Sci. USA 99:11616-11621, whichgenerated a randomized population having a bias comprising a desiredstructure. Therefore a randomized population of modified FRTrecombination sites can be generated to contain a desirable bias in theprimary and/or secondary structure of the site, or various domains ofthe site.

It is not necessary that the library include all possible variantsequences. The library can include as large of a number of possiblesequence variants as is practical for selection, to insure that asufficient number of potential functional modified FRT recombinationsites are identified. For example, if the randomized sequence in themodified FRT recombination site includes the 6 internal spacer residues(see, Table 1), it would contain approximately 4⁶ (or 4096) sequencepermutations using the 4 naturally occurring bases. However, it is notnecessary for the library to include all possible sequences to permitselection of functional modified FRT recombination sites.

Once the members of the population of the randomized modified FRTrecombination sites are generated, the sequences are packaged intoplasmids using standard methods. In some examples, the population ofplasmids can be introduced into suitable cells for both amplificationand storage. Although cloning and amplification are typicallyaccomplished using bacterial cells, any functional combination ofplasmid and cell may be used. The cloned cells can be frozen for futureamplification and use, or the packaged plasmid library can be isolatedand itself stored in any form that preserves viability.

Typical plasmids of interest include vectors having defined cloningsites, origins of replication and selectable markers. The plasmid mayfurther include transcription and translation initiation sequences,transcription and translation terminators, and promoters useful forregulation of the expression of the particular nucleic acid. Plasmidscan also include generic expression cassettes containing at least oneindependent terminator sequence, sequences permitting replication of thecassette in eukaryotes, or prokaryotes, or both, such as shuttlevectors, and selection markers for both prokaryotic and eukaryoticsystems. Vectors may be suitable for replication and integration inprokaryotes, eukaryotes, or both. For general descriptions of cloning,packaging, and expression systems and methods, see Giliman & Smith(1979) Gene 8:81-97; Roberts et al. (1987) Nature 328:731-734; Berger &Kimmel (1989) Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol. 152, Academic Press, Inc., San Diego, Calif., (Berger);Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.)Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press,N.Y., (Sambrook); and F. M. Ausubel et al. (eds.) (1994) CurrentProtocols in Molecular Biology, Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.(1994 Supplement) (Ausubel).

In one example, the members of the population of randomized modified FRTrecombination sites are introduced into a population of plasmids,wherein each of the plasmids in the population comprises a commonselectable marker. In this example, a population of randomized modifiedFRT recombination sites is contacted with a population of plasmids underconditions that allow for the insertion of the population of randomizedmodified FRT recombination sites into each plasmid of the population ofplasmids such that each of the plasmids of said population comprise asingle member of the population of randomized modified FRT recombinationsites. In one example, the selectable marker is operably linked to apromoter active in a host cell of interest. Various selectable markerscan be used in the method.

A selectable or screenable marker comprises a DNA segment that allowsone to identify or select for or against a molecule or a cell thatcontains it, often under particular conditions. Any selectable markercan be used. These markers can encode an activity, such as, but notlimited to, production of RNA, peptide, or protein, or can provide abinding site for RNA, peptides, proteins, inorganic and organiccompounds or compositions and the like. Examples of selectable markersinclude, but are not limited to, DNA segments that comprise restrictionenzyme sites; DNA segments that encode products which provide resistanceagainst otherwise toxic compounds including antibiotics, such as,spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT));DNA segments that encode products which are otherwise lacking in therecipient cell (e.g., tRNA genes, auxotrophic markers); DNA segmentsthat encode products which can be readily identified (e.g., phenotypicmarkers such as β-galactosidase, GUS; fluorescent proteins such as greenfluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cellsurface proteins); the generation of new primer sites for PCR (e.g., thejuxtaposition of two DNA sequence not previously juxtaposed), theinclusion of DNA sequences not acted upon or acted upon by a restrictionendonuclease or other DNA modifying enzyme, chemical, etc.; and, theinclusion of a DNA sequences required for a specific modification (e.g.,methylation) that allows its identification.

Additional selectable markers include genes that confer resistance toherbicidal compounds, such as glufosinate ammonium, bromoxynil,imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally,Yarranton (1992) Curr Opin Biotech 3:506-511; Christopherson et al.(1992) Proc Natl Acad Sci USA 89:6314-6318; Yao et al. (1992) Cell71:63-72; Reznikoff (1992) Mol Microbiol 6:2419-2422; Barkley et al.(1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566;Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell52:713-722; Deuschle et al. (1989) Proc Natl Acad Sci USA 86:5400-5404;Fuerst et al. (1989) Proc Natl Acad Sci USA 86:2549-2553; Deuschle etal. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, Universityof Heidelberg; Reines et al. (1993) Proc Natl Acad Sci USA 90:1917-1921;Labow et al. (1990) Mol Cell Biol 10:3343-3356; Zambretti et al. (1992)Proc Natl Acad Sci USA 89:3952-3956; Baim et al. (1991) Proc Natl AcadSci USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res19:4647-4653; Hillen & Wissman (1989) Topics Mol Struc Biol 10:143-162;Degenkolb et al. (1991) Antimicrob Agents Chemother 35:1591-1595;Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D.Thesis, University of Heidelberg; Gossen et al. (1992) Proc Natl AcadSci USA 89:5547-5551; Oliva et al. (1992) Antimicrob Agents Chemother36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724.

The modified FRT recombination sites, functional modified FRTrecombination sites, and the various populations of such moleculesincluding the libraries and plasmid populations can also be used asreagents in kits. For example, kits that can be employed in the variousmethods disclosed herein are provided. In one example, the kit comprisesa first population of plasmids wherein each of the plasmids in the firstpopulation comprises a first common selectable marker; and, each of theplasmids in the first population comprises a member of a population ofmodified FRT recombination sites. The kit can further include a secondpopulation of plasmids wherein each of the plasmids in said secondpopulation comprises a second common selectable marker, wherein thefirst and the second selectable markers are distinct; and, each of saidplasmids in the second population comprises a member of the populationof modified FRT recombination sites. In other examples, the kits canfurther comprise a FLP recombinase. In still other examples, the kit cancomprise a polynucleotide, optionally integrated in the genome of anorganism, having at least one target site flanked by functional,dissimilar, non-recombinogenic modified FRT recombination site. Any kitcan further be accompanied by instructions for use.

Further provided are kits having a polynucleotide comprising at leastone heterologous functional modified FRT recombination site, saidfunctional modified FRT recombination site comprises a spacer sequenceselected from the group consisting of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17 and 18. Kits having any of the otherpolynucleotides disclosed herein are further provided. In specificexamples, the polynucleotide in the kit further comprises at least oneadditional recombination site. In specific examples, the recombinationsites are dissimilar and non-recombinogenic with respect to one another,dissimilar and recombinogenic with respect to one another, orcorresponding and recombinogenic. Kits can further include one or moreof the appropriate recombinases or a polynucleotide encoding the same.

Populations of plasmids comprising a member of a population of modifiedFRT recombination sites can be generated. Methods to select, identifyand/or characterize modified recombinogenic FRT recombination sites fromthe population of modified FRT recombination sites are provided. In oneexample selection of a recombinogenic FRT recombination site comprisesproviding a first population of plasmids wherein each of the plasmids inthe first population comprises a common first selectable marker; and,each of the plasmids in the first population comprises a heterologousmember of a population of modified FRT recombination sites. A secondpopulation of plasmids is provided. The second population of plasmidscomprises a second common selectable marker, wherein the first and thesecond selectable markers are distinct; and, each of the plasmids in thesecond population comprises a member of the population of modified FRTrecombination sites. A distinct selectable marker indicates that themarker present in the first population of plasmids employs a differentselection scheme or agent than the selectable marker present in thesecond population of plasmids. In other words, the presence of adistinct selectable marker in the two populations of plasmids will allowfor screening of plasmid populations to determine if none, one, or bothof the selectable markers are present.

In one method, the first population of plasmids is combined with thesecond population of plasmids in the presence of a FLP recombinase. Thecomponents are combined in vivo or in vitro under conditions that allowrecombinase-mediated integration to occur. Recombinase-mediatedintegration results in a recombination event between a modifiedrecombinogenic FRT site on one plasmid and a recombinogenic modified FRTsite on a second plasmid. The recombination event results in thegeneration of a co-integrant plasmid. A co-integrant is a nucleic acidmolecule that contains both parental molecules, the plasmid of the firstlibrary and the plasmid of the second library. It will usually becircular, but may also be linear. A co-integrant can comprise plasmidsfrom the first and second library and therefore have two distinctselectable markers. Additional co-integrant plasmids may form betweenplasmids of the same population. These co-integrants comprise commonselectable markers. Selection schemes that allow for the selection ofco-integrants generated via a recombinase-mediated event betweenplasmids from the first and the second plasmid populations are discussedin detail below.

The conditions in which the two populations of plasmids are combinedwill allow the FLP recombinase to mediate a recombination event betweena modified recombinogenic FRT recombination site contained on a plasmidfrom the first population with a modified recombinogenic FRTrecombination site contained on a plasmid from the second population andthereby form a co-integrant plasmid. Conditions that allow for therecombinase mediated integration event can vary. For instance, theamount of recombinase added to drive the recombinase mediatedintegration reaction can be determined using known assays, such astitration assays, to determine the appropriate amount of recombinaseunder given conditions. Similarly, the concentration of both plasmidpopulations can be varied, along with time, temperature and otherreaction conditions to allow for a desired reaction. In one example, theplasmid populations are added in an equimolar ratio.

Any method that allows for the selection, enrichment, or identificationof a co-integrant plasmid can be used in the methods. In one example,the co-integrant will comprise two distinct selectable markers.Accordingly, methods for selecting co-integrants away from the plasmidsthat either failed to undergo a recombinase mediated integration eventor undergo an event between plasmids from the same population can entailintroducing the mixture comprising the co-integrants and the otherunreacted plasmids into a host cell and selecting host cells having bothmarkers.

After the formation of the co-integrant, the selection step can becarried out either in vivo or in vitro depending upon the particularselection scheme being employed, see for example, U.S. Pat. No.6,277,608. The selection schemes that can be employed in the methods andcompositions will vary depending on the selectable marker employed inthe plasmid populations.

In vivo selection schemes can be used with a variety of host cellsincluding, for example, E. coli. A non-limiting example of aco-integrant plasmid along with a non-limiting in vivo selection schemefollows. In this example, plasmid A comprises an ampicillin selectablemarker and a modified FRT site and plasmid B comprise a spectinomycinselectable marker and a corresponding modified FRT recombination site.Upon addition of FLP recombinase, a recombination event between themodified FRT site of plasmid A and plasmid B occurs. The resultingco-integrant plasmid comprises both the ampicillin marker of the plasmidA and the spectinomycin marker of plasmid B. The plasmids from thereaction mixture are introduced into competent E. coli. E. colicontaining co-integrants are resistant to both ampicillin andspectinomycin. Following the selection of co-integrants, the modifiedFRT recombination sites contained on the co-integrant can becharacterized, and the modified FRT recombination sites contained onplasmid A and B can then be determined. For instance, the sites can besequenced. In addition, the recombination excision efficiency can alsobe determined. In some examples the modified FRT site of plasmid A andof plasmid B may also be dissimilar and recombinogenic. In suchinstances, the recombination sites appearing on the co-integrant plasmidmay be sequenced to determine the dissimilar/recombinogenic sitesappearing on plasmid A and plasmid B.

Other schemes for selection include in vitro assays that assay for theselection of the co-integrants through the generation of new primersites for PCR; inclusion of DNA sequences acted upon or not acted uponby a restriction endonuclease or other DNA modifying enzyme, chemical,etc.; selection of the desired product by size or other physicalproperty of the molecule; and inclusion of a DNA sequence required for aspecific modification (e.g., methylation).

Recombinogenic modified FRT recombination sites can be used in variousin vitro and in vivo site-specific recombination methods that allow forthe targeted integration, exchange, modification, alteration, excision,inversion, and/or expression of a nucleotide sequence of interest, seefor example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853.

The methods employ a site-specific recombination system. A site-specificrecombinase, also referred to as a recombinase, is a polypeptide thatcatalyzes conservative site-specific recombination between itscompatible recombination sites, therefore a recombinase includes nativepolypeptides as well as variants and/or fragments that retain activity,and native polynucleotides and variants and/or fragments that encode arecombinase that retains activity. The recombinase used in the methodscan be a native recombinase or a biologically active fragment or variantof the recombinase. A native polypeptide or polynucleotide comprises anaturally occurring amino acid sequence or nucleotide sequence. Forreviews of site-specific recombinases, see Sauer (1994) Curr OpBiotechnol 5:521-527; and Sadowski (1993) FASEB 7:760-767. Recombinasesuseful in the methods and compositions include recombinases from theIntegrase and Resolvase families, biologically active variants andfragments thereof, and any other naturally occurring or recombinantlyproduced enzyme or variant thereof, that catalyzes conservativesite-specific recombination between specified DNA recombination sites.

The Integrase family of recombinases has over one hundred members andincludes, for example, FLP, Cre, lambda integrase, and R. For othermembers of the Integrase family, see for example, Esposito et al. (1997)Nucleic Acids Res 25:3605-3614 and Abremski et al. (1992) Protein Eng5:87-91. Other recombination systems include, for example, thestreptomycete bacteriophage phi C31 (Kuhstoss et al. (1991) J Mol Biol20:897-908); the SSV1 site-specific recombination system from Sulfolobusshibatae (Maskhelishvili et al. (1993) Mol Gen Genet 237:334-342); and aretroviral integrase-based integration system (Tanaka et al. (1998) Gene17:67-76). In other examples, the recombinase is one that does notrequire cofactors or a supercoiled substrate. Such recombinases includethe native Cre (SEQ ID NOS:45 and 46), the native FLP (SEQ ID NOS:48 and49), or active variants or fragments thereof (SEQ ID NOS:47 and 50).

The FLP recombinase is a protein that catalyzes a site-specific reactionthat is involved in amplifying the copy number of the two-micron plasmidof S. cerevisiae during DNA replication. FLP recombinase catalyzessite-specific recombination between two FRT sites. The FLP protein hasbeen cloned and expressed (Cox (1993) Proc Natl Acad Sci USA80:4223-4227). The FLP recombinase for use in the methods and with thecompositions may be derived from the genus Saccharomyces, One can alsosynthesize a polynucleotide comprising the recombinase usingplant-preferred codons for optimal expression in a plant of interest. Arecombinant FLP enzyme encoded by a nucleotide sequence comprising maizepreferred codons (FLPm) that catalyzes site-specific recombinationevents is known (SEQ ID NO:50, and U.S. Pat. No. 5,929,301). Additionalfunctional variants and fragments of FLP are known (Buchholz et al.(1998) Nat Biotechnol 16:617-618, Hartung et al. (1998) J Biol Chem273:22884-22891, Saxena et al. (1997) Biochim Biophys Acta 1340:187-204,and Hartley et al. (1980) Nature 286:860-864).

The bacteriophage recombinase Cre catalyzes site-specific recombinationbetween two lox sites. The Cre recombinase is known (Guo et al. (1997)Nature 389:40-46; Abremski et al. (1984) J Biol Chem 259:1509-1514; Chenet a. (1996) Somat Cell Mol Genet 22:477-488; Shaikh et al. (1977) JBiol Chem 272:5695-5702; and, Buchholz et al. (1998) Nat Biotechnol16:617-618. Cre polynucleotide sequences may also be synthesized usingplant-preferred codons, for example such sequences (moCre) are describedin WO 99/25840 and set forth in SEQ ID NO:47.

It is further recognized that a chimeric recombinase can be used in themethods. A chimeric recombinase is a recombinant fusion protein which iscapable of catalyzing site-specific recombination between recombinationsites that originate from different recombination systems. For exampleif a set of functional recombination sites, characterized as beingdissimilar and non-recombinogenic with respect to one another, isutilized in the methods and compositions, and the set comprises a FRTsite and a LoxP site, a chimeric FLP/Cre recombinase or active variantor fragment thereof will be needed or both recombinases may beseparately provided. Methods for the production and use of such chimericrecombinases or active variants or fragments thereof are described in WO99/25840.

Fragments and variants of the polynucleotides encoding recombinases andfragments and variants of the recombinase proteins are also encompassed.A fragment is a portion of the polynucleotide and/or any protein encodedthereby or a portion of the polypeptide. Fragments of a polynucleotidemay encode protein fragments that retain the biological activity of thenative protein and hence implement a recombination event. Thus,fragments of a polynucleotide may range from at least about 20nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length polynucleotide encoding a recombinase.

A fragment of a polynucleotide that encodes a biologically activeportion of a recombinase protein will encode at least 15, 25, 30, 50,100, 150, 200, 250, 300, 320, 350, 375, 400, or 420 contiguous aminoacids, or up to the total number of amino acids present in a full-lengthrecombinase protein (i.e., 423 amino acids for the FLP recombinase and338 amino acids for the Cre recombinase) used in the methods.

A biologically active portion of a recombinase protein can be preparedby isolating a portion of one of the polynucleotides encoding theportion of the recombinase polypeptide and expressing the encodedportion of the recombinase protein, and assessing the activity of theportion of the recombinase. Polynucleotides that encode fragments of arecombinase polypeptide can comprise nucleotide sequence comprising atleast 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 800, 900, 1,000, 1,100, or 1,200 nucleotides, or up tothe number of nucleotides present in a full-length recombinasenucleotide sequence (i.e., 1032 nucleotides for the FLP recombinase and1260 nucleotides for the Cre recombinase) disclosed herein.

Variant sequences have a high degree of sequence similarity. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the native recombinase polypeptides. Variants such asthese can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR) andhybridization techniques. Variant polynucleotides also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode arecombinase protein. Generally, variants of a particular polynucleotidewill have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to that particular polynucleotide as determined by knownsequence alignment programs and parameters.

Variants of a particular polynucleotide (the reference nucleotidesequence) can also be evaluated by comparison of the percent sequenceidentity between the polypeptide encoded by a variant polynucleotide andthe polypeptide encoded by the reference polynucleotide. Thus, forexample, isolated polynucleotides that encode a polypeptide with a givenpercent sequence identity to the recombinase are known. Percent sequenceidentity between any two polypeptides can be calculated using sequencealignment programs and parameters described. Where any given pair ofpolynucleotides is evaluated by comparison of the percent sequenceidentity shared by the two polypeptides they encode, the percentsequence identity between the two encoded polypeptides is at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

A variant protein is intended a protein derived from the native proteinby deletion, addition, and/or substitution of one or more amino acids tothe N-terminal, internal region(s), and/or C-terminal end of the nativeprotein. Variant proteins are biologically active, that is they continueto possess the desired biological activity of the native protein, forexample a variant recombinase will implement a recombination eventbetween appropriate recombination sites. Such variants may result from,for example, genetic polymorphism or from human manipulation.Biologically active variants of a native recombinase protein will haveat least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity tothe amino acid sequence for the native protein as determined by knownsequence alignment programs and parameters. A biologically activevariant of a protein may differ from that protein by as few as 1-15amino acid residues, as few as 1-10, such as 6-10, as few as 5, as fewas 4, 3, 2, or even 1 amino acid residue.

Sequence relationships can be analyzed and described usingcomputer-implemented algorithms. The sequence relationship between twoor more polynucleotides, or two or more polypeptides can be determinedby generating the best alignment of the sequences, and scoring thematches and the gaps in the alignment, which yields the percent sequenceidentity, and the percent sequence similarity. Polynucleotiderelationships can also be described based on a comparison of thepolypeptides each encodes. Many programs and algorithms for thecomparison and analysis of sequences are available.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 (GCG, Accelrys,San Diego, Calif.) using the following parameters: % identity and %similarity for a nucleotide sequence using a gap creation penalty weightof 50 and a gap length extension penalty weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using a GAP creation penalty weight of 8 and a gap lengthextension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff &Henikoff (1989) Proc Natl Acad Sci USA 89:10915).

GAP uses the algorithm of Needleman & Wunsch (1970) J Mol Biol48:443-453, to find an alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. GAP presents one member of the family of bestalignments.

Sequence identity, or identity, is a measure of the residues in the twosequences that are the same when aligned for maximum correspondence.Sequences, particularly polypeptides, that differ by conservativesubstitutions are said to have sequence similarity or similarity. Meansfor making this adjustment are known, and typically involve scoring aconservative substitution as a partial rather than a full mismatch. Forexample, where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions is calculated using the selected scoringmatrix (BLOSUM62 by default for GAP).

Proteins may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known. For example, amino acid sequencevariants of the recombinase proteins can be prepared by mutations in theDNA. Methods for mutagenesis and nucleotide sequence alterations includefor example, Kunkel (1985) Proc Natl Acad Sci USA 82:488-492; Kunkel etal. (1987) Methods in Enzymol 154:367-382; U.S. Pat. No. 4,873,192;Walker & Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl Biomed ResFound, Washington, D.C.). Conservative substitutions, such as exchangingone amino acid with another having similar properties, may bepreferable.

The recombinase polynucleotides used include both the naturallyoccurring native sequences as well as mutant or modified forms.Likewise, the proteins used in the methods encompass both naturallyoccurring proteins as well as variations and modified forms thereof.Such variants continue to possess the ability to implement arecombination event. Generally, the mutations made in the polynucleotideencoding the variant polypeptide do not place the sequence out ofreading frame or create complementary regions that could producesecondary mRNA structure. See, EP Patent Application Publication No.75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, the effect will be evaluated by routine screening assays.Assays for recombinase activity are known and generally measure theoverall activity of the enzyme on DNA substrates containingrecombination sites. For example, to assay for FLP activity, inversionof a DNA sequence in a circular plasmid containing two inverted FRTsites can be detected as a change in position of restriction enzymesites. This assay is described in Vetter et al. (1983) PNAS 80:7284.Alternatively, excision of DNA from a molecule or intermolecularrecombination frequency induced by the enzyme may be assayed, asdescribed, for example, in Babineau et al. (1985) J Biol Chem 260:12313;Meyer-Leon et al. (1987) Nucleic Acid Res 15:6469; and Gronostajski etal. (1985) J Biol Chem 260:12328. Alternatively, recombinase activitymay also be assayed by excision of a sequence flanked by recombinogenicFRT sites that upon removal will activate an assayable marker gene.Similar assay strategies may be used for Cre or other recombinaseenzymes.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and/or recombinogenic procedure suchas DNA shuffling. With such a procedure, one or more differentrecombinase coding sequences can be manipulated to create a newrecombinase protein possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known andinclude for example, Stemmer (1994) Proc Natl Acad Sci USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nat Biotech 15:436-438; Moore et al. (1997) J Mol Biol 272:336-347;Zhang et al. (1997) Proc Natl Acad Sci USA 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The methods and compositions can further employ recombination sitesother than the modified FRT sites provided herein. A recombination siteis any native or synthetic/artificial polynucleotide that is recognizedby the recombinase enzyme of interest. Many recombination systems areknown as is the appropriate recombination site(s) to be used with therecombination system of interest, including biologically active variantsand fragments of recombination sites. Examples of recombination sitesfor use are known and include FRT sites including the native FRT site(FRT1, SEQ ID NO:39), and various functional variants of FRT, includingbut not limited to, FRT5 (SEQ ID NO:40), FRT6 (SEQ ID NO:41), FRT7 (SEQID NO:42), FRT87 (SEQ ID NO:24), and the other functional modified FRTsites disclosed herein. See, for example, WO 03/054189, WO 02/00900, WO01/23545, and, Schlake et al. (1994) Biochemistry 33:12745-12751

Recombination sites from the Cre/Lox site-specific recombination systemcan also be used. Such recombination sites include, for example, nativeLOX sites and various functional variants of LOX. An analysis of therecombination activity of variant LOX sites is presented in Lee et al.(1998) Gene 216:55-65 and in U.S. Pat. No. 6,465,254. Also, see forexample, Schlake & Bode (1994) Biochemistry 33:12746-12751; Huang et al.(1991) Nucleic Acids Res 19:443-448; Sadowski (1995) In Progress inNucleic Acid Research and Molecular Biology Vol. 51, pp. 53-91; U.S.Pat. No. 6,465,254; Cox (1989) In Mobile DNA, Berg and Howe (eds)American Society of Microbiology, Washington D.C., pp. 116-670; Dixon etal. (1995) Mol Microbiol 18:449-458; Umlauf & Cox (1988) EMBO J7:1845-1852; Buchholz et al. (1996) Nucleic Acids Res 24:3118-3119;Kilby et al. (1993) Trends Genet 9:413-421; Rossant & Geagy (1995) NatMed 1:592-594; Albert et al. (1995) Plant J 7:649-659; Bayley et al.(1992) Plant Mol Biol 18:353-361; Odell et al. (1990) Mol Gen Genet223:369-378; Dale & Ow (1991) Proc Natl Acad Sci USA 88:10558-10562; Quiet al. (1994) Proc Natl Acad Sci USA 91:1706-1710; Stuurman et al.(1996) Plant Mol Biol 32:901-913; Dale et al. (1990) Gene 91:79-85; andWO 01/111058.

Any suitable recombination site or set of recombination sites may beutilized in the methods and compositions, including a FRT site, afunctional variant of a FRT site, a LOX site, and functional variant ofa LOX site, any combination thereof, or any other combination ofrecombination sites known.

Directly repeated indicates that the recombination sites in a set ofrecombinogenic recombination sites are arranged in the same orientation,such that recombination between these sites results in excision, ratherthan inversion, of the intervening DNA sequence. Inverted recombinationsite(s) indicates that the recombination sites in a set ofrecombinogenic recombination sites are arranged in the oppositeorientation, so that recombination between these sites results ininversion, rather than excision, of the intervening DNA sequence.

The target site and transfer cassette comprise at least onerecombination site. The site-specific recombinase(s) used will dependupon the recombination sites present in the target site and the transfercassette, for example if FRT sites are utilized, a FLP recombinase oractive variant thereof will be provided. In the same manner, where Loxsites are utilized, a Cre recombinase or active variant thereof isprovided. If the set of functional recombination sites comprises both aFRT site and a Lox site, either a chimeric FLP/Cre recombinase or anactive variant or both FLP and Cre recombinases or active variantsthereof will be provided. In one example, at least one of therecombination sites employed at the target site, transfer cassette, orboth will comprise at least one functional modified FRT recombinationsite disclosed herein.

Providing includes any method that allows for a polypeptide and/or apolynucleotide such as a recombinase, target site, transfer cassette,polynucleotide of interest to be brought together with the recitedcomponents. For instance, a cell can be provided with these variouscomponents via a variety of methods including transient and stabletransformation methods; co-introducing a recombinase DNA, mRNA orprotein directly into the cell; employing an organism, cell, strain orline that expresses the recombinase for the initial transformation; orgrowing/culturing the organism carrying the target site and crossing itto an organism that expresses an active recombinase protein andselecting events in the progeny. Any promoter including constitutive,inducible, developmentally/temporal, or spatially regulated promotercapable of regulating expression in the organism of interest may be usedto express the appropriate recombinase.

Compositions comprising recombinogenic modified FRT recombination sitesare provided, along with biologically active variants and fragments ofthe recombinogenic modified FRT recombinant sites. The recombinogenicmodified FRT recombination site can be used in various site-specificrecombination methods.

The methods can employ target sites and transfer cassettes to allow forthe manipulation, exchange, excision, alteration, inversion and/orintroduction of a nucleotide sequence in vivo or in vitro. A target sitecomprises at least one recombination site. In specific examples, thetarget site comprises a polynucleotide that is immediately flanked by atleast two recombination sites, including sets of functionalrecombination sites that are dissimilar and non-recombinogenic withrespect to one another; corresponding and recombinogenic with respect toone another; or dissimilar and recombinogenic with respect to oneanother. One or more intervening sequences may be present between therecombination sites of the target site. Intervening sequences ofparticular interest include linkers, adapters, regulatory regions,introns, restriction sites, enhancers, insulators, selectable markers,nucleotide sequences of interest, promoters, and/or other sites that aidin vector construction or analysis. It is further recognized that arecombination site can be contained within the nucleotide sequence ofinterest including introns, coding sequence, 5′ UTRs, 3′ UTRs, and/orregulatory regions.

In specific examples, the target site is in a cell or an organism ofinterest. In other examples, the target site is stably integrated intothe genome of the cell or the organism of interest. It is recognizedthat the cell or the organism may comprise multiple target sites, whichmay be located at one or multiple loci within or across chromosomes.Multiple independent manipulations of each target site in the organismare available. Additionally, the target site may also comprise anexpression cassette comprising a nucleotide sequence encoding anappropriate recombinase. In another example, the nucleotide sequenceencoding the recombinase is stably integrated in the genome of theorganism.

The methods further employ transfer cassettes. A transfer cassettecomprises at least one recombination site. In specific examples, thetransfer cassette comprising a polynucleotide flanked by at least afirst recombination site and a second recombination site, wherein thefirst and second recombination sites correspond to the recombinationsites in the target site. The first and the second functionalrecombination sites of the transfer cassette can be dissimilar andnon-recombinogenic with respect to one another. When a target site and atransfer cassette comprising compatible recombination sites and therecombinase are combined the nucleotide sequence between therecombination sites of the target site will be exchanged with thenucleotide sequence between the recombination sites of the transfercassette. Flanked by, when used in reference to the position of therecombination sites of the target site or the transfer cassette, refersto a position immediately adjacent to the sequence intended to beexchanged or inserted.

The transfer cassette can further comprise a polynucleotide of interest.The recombination sites may be directly contiguous with thepolynucleotide of interest or there may be one or more interveningsequences present between one or both ends of the polynucleotide ofinterest and the recombination sites. Intervening sequences ofparticular interest include linkers, adapters, enhancers, introns,insulators, restriction sites, selectable markers, polynucleotides ofinterest, promoters, and/or other sites that aid in vector constructionor analysis. The recombination sites can be contained within thepolynucleotide of interest including within introns, coding sequence,and/or 5′ and 3′ untranslated regions.

In one example, the transfer cassette and/or the target site comprise atleast one functional modified FRT recombination site, where thefunctional modified FRT recombination site comprises a spacer sequenceselected from the group consisting of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. In other examples, thetransfer cassette and/or target site comprises at least one functionalmodified FRT recombination site comprising SEQ ID NO:21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or a functionalvariant thereof. The functional variant can comprise one, two, three,four, five, six, or more alterations between nucleotide positions 1 to11 and/or between nucleotide positions 20 to 30 of SEQ ID NO:21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38. In stillother examples, the functional variant is substantially identical to thesequence of SEQ ID NOS:21-38.

Any means can be used to bring together the various components of therecombination system. For example, in in vitro systems, the recombinaseand the polynucleotide(s) comprising the recombination sites can beprovided by contacting the components under the appropriate conditionsto allow for a recombination event. Alternatively a variety of methodsare known for the introduction of nucleotide sequences and polypeptidesinto an organism, including, for example, transformation, sexualcrossing, and the introduction of the polypeptide, DNA, or mRNA into thecell. See, also, WO99/25884.

The methods find use in various applications. For example, the methodscan employ the use of two modified functional FRT recombination sitesand allow for in vivo and in vitro exchange, insertion, inversion, orexcision of a nucleotide sequence of interest. For example, the cell orthe organism of interest can comprise a first polynucleotide comprisinga target site comprising a first functional modified FRT recombinationsite. The cell or the organism is provided a second polynucleotidecomprising a transfer cassette comprising either a second correspondingand functional FRT recombination site or a second dissimilar FRT sitethat is recombinogenic with respect to the first site. A FLP recombinaseis provided under conditions that allow for a recombination event. Therecombination event between the two recombinogenic recombination sitesresults in the insertion of the transfer cassette along with the entiresecond polynucleotide it is contained on into the first polynucleotide.In some examples, the first polynucleotide is stably integrated into thegenome of the organism. The method can also be employed in an in vitrocontext. For example, the first and the second polynucleotides cancomprise polynucleotides such as plasmids combined in vitro in thepresence of an appropriate recombinase. In this example, a recombinationevent will produce a co-integrant plasmid. Such methods find use, forexample, in various cloning technologies, including PCR-amplification offragments (Sadowski et al. (2003) BMC Biotechnol 18:9), cloning vectors(Snaith et al. (1995) Gene 166:173-174 and U.S. Pat. Nos. 6,140,129,6,410,317, 6,355,412, 5,888,732, 6,143,557, 6,171,861, 6,270,969, and6,277,608) and viral vectors (U.S. Pat. No. 6,541,245).

In other examples, the method comprises providing a target site having afirst and a second functional recombination site, wherein the first andthe second recombination sites are dissimilar and non-recombinogenicwith respect to one another and at least one of the first or the secondrecombination sites comprise a functional modified FRT recombinationsite disclosed herein; providing a transfer cassette comprising apolynucleotide of interest flanked by the first and the secondrecombination site; and, providing a recombinase. The recombinaserecognizes and implements recombination at the first and the secondrecombination sites.

In specific examples, the target site is in a cell or host organism;and, in other examples, the target site is stably integrated into thegenome of the cell or the host organism. In still other examples, thetarget site comprises a polynucleotide of interest. In this example, ifthe target site and transfer cassette comprise the first and the secondrecombination sites which are dissimilar and non-recombinogenic withrespect to one another the sequence of interest in the target site isexchanged for a second polynucleotide of interest contained in thetransfer cassette.

In other examples, the compositions provided herein are used in methodsto reduce the complexity of integration of transgenes in the genome of acell or an organism, such as a plant cell or a plant. In this method,organisms having simple integration patterns in their genomes areselected. A simple integration pattern indicates that the transfercassette integrates predominantly only at the target site, and at lessthan about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 randomposition(s) other than the target site in the genome. Methods fordetermining the integration patterns are known in the art and include,for example, Southern blot analysis and RFLP analysis.

A method to directly select a transformed cell or an organism, such as aplant or plant cell is provided. The method comprises providing apopulation of cells or organisms having a polynucleotide comprising atarget site. The polynucleotide comprises, in the following order, apromoter and a target site comprising a first and a second functionalrecombination site, wherein the first and the second recombination sitesare dissimilar and non-recombinogenic with respect to one another and atleast one of the first or the second recombination sites comprise afunctional modified FRT recombination site provided herein. A transfercassette is introduced into the population of cells or organisms, wherethe transfer cassette comprises, in the following order, the firstrecombination site, a polynucleotide comprising a selectable marker notoperably linked to a promoter, and the second recombination site. Arecombinase or a biologically active fragment is provided thatrecognizes and implements recombination at the first and secondrecombination sites, and thereby operably linking the selectable markerto the promoter. The population of cells or organisms is then grown onthe appropriate selective agent to recover the organism that hassuccessfully undergone targeted integration of the transfer cassette atthe target site. In other examples, the population of cells or organismshas stably incorporated into their genomes the target site.

The activity of various promoters at a characterized location in thegenome of a cell or an organism can be determined. Thus, the desiredactivity and/or expression level of a nucleotide sequence of interestcan be achieved, as well as, the characterization of promoters forexpression in the cell or the organism of interest. In one example, themethod for assessing promoter activity in a cell or an organismcomprises providing a cell or an organism comprising in its genome apolynucleotide comprising a target site having a first and a secondfunctional recombination site, wherein the first and the secondrecombination sites are dissimilar and non-recombinogenic with respectto one another, wherein at least one of the first or the secondfunctional recombination sites comprises a functional modified FRTrecombination site provided herein. A transfer cassette is introducedinto the cell or the organism, where the transfer cassette comprises apromoter operably linked to a polynucleotide comprising a selectablemarker and the transfer cassette is flanked by the first and the secondfunctional recombination sites. A recombinase is provided, wherein saidrecombinase recognizes and implements recombination at the first andsecond recombination sites. Promoter activity is assessed by monitoringexpression of the selectable marker. In this manner, different promoterscan be integrated at the same position in the genome and their activitycompared.

In some examples, the transfer cassette comprises in the followingorder: the first recombination site, a promoter operably linked to athird recombination site operably linked to a polynucleotide comprisinga selectable marker, and the second recombination site, where the first,the second, and the third functional recombination sites are dissimilarand non-recombinogenic with respect to one another. This transfercassette can be generically represented as RSa-P1::RSc::S1-RSb.Following the introduction of the transfer cassette at the target site,the activity of the promoter (P1) can be analyzed using methods known inthe art. Once the activity of the promoter is characterized, additionaltransfer cassettes comprising a polynucleotide of interest flanked bythe second and the third recombination site can be introduced into theorganism. Upon recombination, the expression of the polynucleotide ofinterest will be regulated by the characterized promoter. Accordingly,organisms, such as plant lines, having promoters that achieve thedesired expression levels in the desired tissues can be engineered sothat nucleotide sequences of interest can be readily inserted downstreamof the promoter and operably linked to the promoter and therebyexpressed in a predictable manner.

In some examples multiple promoters can be employed to regulatetranscription at a single target site. In this method, the target sitecomprising the first and the second recombination sites is flanked bytwo convergent promoters. Convergent promoters refers to promoters thatare oriented on either terminus of the target site. The same promoter,or different promoters may be used at the target site. Each of theconvergent promoters is operably linked to either the first or thesecond recombination site. For example, the target site flanked by theconvergent promoters can comprise P1→R1-R2←P2, where P is a promoter,the arrow indicates the direction of transcription, R is a recombinationsite, and the colon indicates the components are operably linked.

The transfer cassette employed with the target site having theconvergent promoters can comprise, in the following order, the firstrecombination site, a first polynucleotide of interest orientated in the5′ to 3′ direction, a second polynucleotide of interest orientated inthe 3′ to 5′ direction, and a second recombination site. The insertionof the transfer cassette at the target site results in the firstpolynucleotide of interest operably linked to the first convergentpromoter, and the second polynucleotide of interest operably linked tothe second convergent promoter. The expression of the first and/or thesecond polynucleotide of interest may be increased or decreased in thecell or organism. The expression of the first and/or the secondpolynucleotide of interest may also be independently regulated dependingupon which promoters are used. It is recognized that target sites can beflanked by other elements that influence transcription. For example,insulator elements can flank the target site to minimize positioneffects. See, for example, U.S. Publication No. 2005/0144665.

Any promoter can be used, and is typically selected based on the desiredoutcome. A promoter is a region of DNA involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A plant promoter is a promoter capable of initiating transcription in aplant cell, for a review of plant promoters see Potenza et al. (2004) InVitro Cell Dev Biol 40:1-22.

Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol Biol12:619-632 and Christensen et al. (1992) Plant Mol Biol 18:675-689);pEMU (Last et al. (1991) Theor Appl Genet 81:581-588); MAS (Velten etal. (1984) EMBO J 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters are described in, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

In some examples an inducible promoter may be used. Pathogen-induciblepromoters induced following infection by a pathogen include, but are notlimited to those regulating expression of PR proteins, SAR proteins,beta-1,3-glucanase, chitinase, etc. See, for example, Redotfi et al.(1983) Neth J Plant Pathol 89:245-254; Uknes et al. (1992) Plant Cell4:645-656; Van Loon (1985) Plant Mol Virol 4:111-116; WO 99143819;Marineau et al. (1987) Plant Mol Biol 9:335-342; Matton et al. (1989)Mol Plant-Microbe Interact 2:325-331; Somsisch et al. (1986) Proc NatlAcad Sci USA 83:2427-2430; Somsisch et al. (1988) Mol Gen Genet 2:93-98;Yang (1996) Proc Natl Acad Sci USA 93:14972-14977; Chen et al. (1996)Plant J 10:955-966; Zhang et al. (1994) Proc Natl Acad Sci USA91:2507-2511; Warner et al. (1993) Plant J 3:191-201; Siebertz et al.(1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein; and Cordero etal (1992) Physiol Mol Plant Path 41:189-200(Fusarium-inducible).Wound-inducible promoters include potato proteinase inhibitor (pin II)gene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan et al. (1996) NatBiotechnol 14:494-498); wun1 and wun2 (U.S. Pat. No. 5,428,148); win1and win2 (Stanford et al. (1989) Mol Gen Genet 215:200-208); systemin(McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al.(1993) Plant Mol Biol 22:783-792; Eckelkamp et al. (1993) FEBS Lett323:73-76); MPI gene (Corderok et al. (1994) Plant J 6:141-150); and thelike. Chemical-regulated promoters can be used to modulate theexpression of a gene in a plant through the application of an exogenouschemical regulator. The promoter may be a chemical-inducible promoter,where application of the chemical induces gene expression, or achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical-inducible promoters include, but arenot limited to, the maize In2-2 promoter, activated bybenzenesulfonamide herbicide safeners (De Veylder et al. (1997) PlantCell Physiol 38:568-77), the maize GST promoter (GST-II-27, WO93101294), activated by hydrophobic electrophilic compounds used aspre-emergent herbicides, and the tobacco PR-la promoter (Ono et al.(2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid.Other chemical-regulated promoters of interest includesteroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena et al. (1991) Proc Natl AcadSci USA 88:10421-10425; and McNellis et al. (1998) Plant J 14:247-257);tetracycline-inducible and tetracycline-repressible promoters (Gatz etal. (1991) Mol Gen Genet 227:229-237; U.S. Pat. Nos. 5,814,618, and5,789,156).

Tissue-preferred promoters can be utilized to target enhanced expressionof a sequence of interest within a particular plant tissue.Tissue-preferred promoters include Kawamata et al. (1997) Plant CellPhysiol 38:792-803; Hansen et al. (1997) Mol Gen Genet 254:337-343;Russell et al. (1997) Transgenic Res 6:157-168; Rinehart et a. (1996)Plant Physiol 112:1331-1341; Van Camp et al. (1996) Plant Physiol112:525-535; Canevascini et al. (1996) Plant Physiol 112:513-524; Lam(1994) Results Probl Cell Differ 20:181-196; and Guevara-Garcia et al.(1993) Plant J 4:495-505.

Leaf-preferred promoters are known and include, for example, Yamamoto etal. (1997) Plant J 12:255-265; Kwon et al. (1994) Plant Physiol105:357-67; Yamamoto et al. (1994) Plant Cell Physiol 35:773-778; Gotoret al. (1993) Plant J 3:509-18; Orozco et al. (1993) Plant Mol Biol23:1129-1138; Matsuoka et al. (1993) Proc Natl Acad Sci USA90(20):9586-9590; and cab and rubisco promoters (Simpson et al. (1958)EMBO J 4:2723-2729; Timko et al. (1988) Nature 318:57-58).

Root-preferred promoters are known and include, for example, Hire et al.(1992) Plant Mol Biol 20:207-218 (soybean root-specific glutaminesynthase gene); Miao et al. (1991) Plant Cell 3:11-22 (cytosolicglutamine synthase (GS) expressed in roots and root nodules of soybean;Keller & Baumgartner (1991) Plant Cell 3:1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol Biol 14:433-443 (root-specific promoter of A.tumefaciens mannopine synthase (MAS)); Bogusz et al. (1990) Plant Cell2:633-641 (root-specific promoters isolated from Parasponia andersoniiand Trema tomentosa); Leach & Aoyagi (1991) Plant Sci 79:69-76 (A.rhizogenes rolC and rolD root-inducing genes); Teeri et al. (1989) EMBOJ 8:343-350 (Agrobacterium wound-induced TR1′ and TR2′ genes);VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol Biol29:759-772); and rolB promoter (Capana et a. (1994) Plant Mol Biol25(4):681-691; phaseolin gene (Murai et al. (1983) Science 23:476-482;Sengopta-Gopalen et al. (1988) Proc Natl Acad Sci USA 82:3320-3324). Seealso U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252;5,401,836; 5,110,732; and 5,023,179.

Seed-preferred promoters include both seed-specific promoters activeduring seed development, as well as seed-germinating promoters activeduring seed germination. See Thompson et al. (1989) BioEssays 10:108.Seed-preferred promoters include, but are not limited to, Cim1(cytokinin-induced message); cZ1981 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase); (see WO 00/11177 and U.S. Pat. No.6,225,529). For dicots, seed-preferred promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-preferred promotersinclude, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDagamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, and nuc1.See also WO 00/12733, where seed-preferred promoters from end1 and end2genes are disclosed.

In further examples, methods are provided to identify a cistranscriptional regulatory region in an organism. A transcriptionalregulatory region is any cis acting element that modulates the level ofan RNA. Such elements include, but are not limited to, a promoter, anelement of a promoter, an enhancer, an insulator, an intron, or aterminator region that is capable of modulating the level of RNA in acell. The methods can be used to generate enhancer or promoter traps. Inone example, the reporter or marker gene of the target site is expressedonly when it inserts close to (enhancer trap) or within (promoter trap)another gene. The expression pattern of the reporter gene will depend onthe enhancer elements of the gene near or in which the reporter geneinserts. In this example, the target site introduced into the cell orthe organism can comprise a marker gene operably linked to arecombination site. In specific examples, the marker gene is flanked bydissimilar and non-recombinogenic recombination sites. The marker geneis either not operably linked to a promoter (promoter trap) or themarker gene is operably linked to a promoter that lacks enhancerelements (enhancer trap). Following insertion of the target site intothe genome of the cell or the organism, the expression pattern of themarker gene is determined for each transformant. When a transformantwith a marker gene expression pattern of interest is found, theenhancer/promoter trap sequences can be used as a probe to clone thegene that has that expression pattern, or alternatively to identify thepromoter or enhancer regulating the expression. In addition, once atarget site is integrated and under transcriptional control of atranscriptional regulatory element, the methods can further be employedto introduce a transfer cassette having a polynucleotide of interestinto that target in the cell or the organism. A recombination eventbetween the target site and the transfer cassette will allow thenucleotide sequence of interest to come under the transcriptionalcontrol of the promoter and/or enhancer element. See, for example,Geisler et a (2002) Plant Physiol 130:1747-1753; Topping et al. (1997)Plant Cell 10:1713-245; Friedrich et al. (1991) Genes Dev 5:1513-23;Dunn et al. (2003) Appl Environ Microbiol 1197-1205; and von Melchner etal. (1992) Genes Dev 6:919-27.

In other examples, the target site is constructed to have multiplefunctional sets of dissimilar and non-recombinogenic recombinationsites. Thus, multiple genes or polynucleotides can be stacked orordered. In specific examples, this method allows for the stacking ofsequences of interest at precise locations in the genome of a cell or anorganism. Likewise, once a target site has been established within acell or an organism, additional recombination sites may be introduced byincorporating such sites within the transfer cassette. Thus, once atarget site has been established, it is possible to subsequently addsites or alter sites through recombination. Such methods are describedin detail in WO 99/25821.

In one example, methods to combine multiple transfer cassettes areprovided. The method comprises providing a target site comprising atleast a first and a second functional recombination site, wherein thefirst and the second recombination sites are dissimilar andnon-recombinogenic with respect to one another. A first transfercassette comprising in the following order at least the first, a third,and the second functional recombination sites is provided wherein thefirst and the third recombination sites of the first transfer cassetteflank a first polynucleotide of interest and wherein the first, thesecond, and the third recombination sites are dissimilar andnon-recombinogenic with respect to one another and a first recombinaseis provided, whereby the first transfer cassette is integrated at thetarget site. At least one of the first, the second, or the thirdrecombination sites comprise a functional modified FRT recombinationsite provided herein.

A second transfer cassette comprising at least the second and the thirdrecombination site is provided, wherein the second and the thirdrecombination sites of the second transfer cassette flank a secondpolynucleotide of interest and a second recombinase is provided. Thesecond recombinase recognizes and implements recombination at the secondand third recombination sites and the second transfer cassettes isinserted at the target site, so that now the first and the secondtransfer cassette are now combined at the target site. In some examples,the target site is in an organism. In other examples, the target site isstably incorporated into the genome of the organism, for example aplant. In this example, multiple polynucleotides of interest are stackedat a predetermined target position in the genome of the organism.

Various alterations can be made to the stacking method to achieve thedesired outcome of having the nucleotides sequences of interest stacked.For instance, a target site comprising in the following order at least afirst, a second, and a third functional recombination site, wherein therecombination sites are dissimilar and non-recombinogenic with respectto one another is provided. A first transfer cassette comprising atleast the first and the second recombination sites is provided, whereinthe first and the second recombination site of the first transfercassette flank a first polynucleotide of interest and a firstrecombinase is provided and recognizes and implements recombination atthe first and the second recombination sites. At least one of the first,the second, or the third recombination sites comprise a functionalmodified FRT recombination site provided herein. A second transfercassette comprising at least the second and the third recombinationsites is provided, where the second and the third recombination sites ofthe second transfer cassette flank a second polynucleotide of interest.A second recombinase is provided. The recombinase recognizes andimplements recombination at the second and third recombination sites.

In other examples methods are provided to minimize or eliminateexpression resulting from random integration of DNA sequences into thegenome of a cell or an organism, such as a plant. This method comprisesproviding a cell or an organism having stably incorporated into itsgenome a polynucleotide comprising the following components in thefollowing order: a promoter active in the cell or the organism operablylinked to an ATG translational start sequence operably linked to atarget site comprising a first and a second functional recombinationsite, wherein the first and the second recombination sites aredissimilar and non-recombinogenic with respect to one another, and atleast one of the first or the second recombination sites comprise afunctional modified FRT recombination site provided herein. A transfercassette comprising a polynucleotide of interest and the first and thesecond recombination site is introduced into the cell or the organism.The translational start sequence of the nucleotide sequence of interestin the transfer cassette has been replaced with the first recombinationsite. A recombinase is provided that recognizes and implementsrecombination at the recombination sites. Recombination with the targetsite results in the polynucleotide of interest being operably linked tothe ATG translational start site of the target site contained in thepolynucleotide. Operably linked indicates a functional fusion betweenadjacent elements, for example the linkage between a translationalstart, a promoter, and/or a recombination site indicates that thesequences are put together to generate an in-frame fusion that resultsin a properly expressed and functional gene product.

In one example a transfer cassette comprising RSc::S3(noATG)-RSd, whereRS represents a recombination site and S represents a polynucleotide ofinterest, is introduced into a plant having stably incorporated into itsgenome a polynucleotide comprising P1-RSa-S1-T1-RSb-P2-ATG::RSc-S2(noATG)-T2-RSd, where P represents a promoter, T represents a terminator,RS represents a recombination site, and the symbol :: indicates operablylinked adjacent elements. ATG::RS indicates that the sequences generatean in-frame fusion that results in a property expressed and functionalgene product. An appropriate recombinase is provided and recombinationtakes place at the recombination sites such that the sequence betweenthe recombination sites of the transfer cassette replaces the sequencebetween the recombination sites of the target site, thereby yielding adirectionally targeted and reintegrated new sequence. The new gene (S3)is now driven off of the P2 promoter in the target site. Designingconstructs without an ATG start codon on the nucleotide sequence ofinterest results in an extremely low probability of expression of theintroduced sequence if random integration occurs, since the transfercassette would need to integrate behind an endogenous promoter regionand in the correct reading frame.

The FRT recombination sites provided herein can be used to excise orinvert a polynucleotide of interest. In this method, a polynucleotide isproviding comprising, in the following order, a first functionalrecombination site, a polynucleotide of interest, and a secondfunctional recombination site, where the first and the secondrecombination sites are recombinogenic with respect to one another.Depending on the orientation of the recombination sites, thepolynucleotide of interest will be excised or inverted when theappropriate recombinase is provided. For example, directly repeatedrecombination sites will allow for excision of the polynucleotide ofinterest and inverted repeats will allow for an inversion of thepolynucleotide of interest. Such methods can be employed either in vivoor in vitro.

Methods are also provided for the alteration of the recombination sites.The method provides for converting between different recombination sitesand is based on previously described oligonucleotide mediated strategiesfor making specific targeted nucleotide modifications at a specifiedextrachromosomal or genomic target sequence (Yoon et al. (1996) ProcNatl Acad Sci USA 93:2071-2076; Cole-Strauss et al. (1996) Science273:1386-1389; WO99125853; WO99/25821; and WO 03/076574). Using thesemethods, the recombination sites can be targeted and modified in variousways. For example, a recombination site could be modified such that thefunctional pair of dissimilar and non-recombinogenic recombination sitesare altered to generate two corresponding and recombinogenicrecombination sites. Subsequent or concurrent expression of theappropriate recombinase in cells with the modified,corresponding/recombinogenic sites would lead to excision or inversionof the sequences between these new recombination sites, depending on theorientation of the sites, thereby specifically removing or turning offexpression of the undesirable DNA sequences from the previously createdconstruct containing these sequences. A non-limiting application of thisapproach would be, for example, in the case of a selectable marker whichis required during initial steps of a breeding or backcrossing programto maintain and select for preferred individual plants, but which is notdesired in the final product. Various oligonucleotide molecules fortargeted modification of recombination sites can be designed and willvary depending on the recombination site being targeted. Exemplaryoligonucleotides designed to modify recombination sites are described inWO99/25821 and WO 03/076574.

Recombination site conversion can also be employed in the methods tostack various polynucleotides in the genome of an organism, such as aplant. For example, the capabilities of the system can be extended by invivo conversion of recombination sites to create new sites, rather thanre-introducing new recombination sites into the organism. For example,conversion of dissimilar and non-recombinogenic recombination sitesflanking a selectable marker to corresponding recombination sites wouldfacilitate removal of a selectable marker, or to allow re-use of thesame selectable marker in future transformations, providing a means torecycle selectable markers. A dissimilar recombination site with a knownrecombination frequency could also be modified in situ to a differentrecombination site with a similar or altered recombination frequency.Other modifications to alter the function, similarity, orrecombinogencity can be accomplished.

In other examples, methods for locating preferred integration siteswithin the genome of a cell or an organism are provided. The methodcomprises introducing into a cell or an organism a target sitecomprising a nucleotide sequence operably linked to a promoter active inthe organism. In specific examples, the target site is flanked by afirst and a second functional recombination site, wherein the first andthe second recombination sites are dissimilar and non-recombinogenicwith respect to one another and at least one of the first or the secondrecombination sites comprises a modified FRT site provided herein. Thelevel of expression of the polynucleotide is determined and the organismexpressing the polynucleotide is selected. The cell or the organismharboring this DNA construct can then be characterized for site-specificintegration potential, agronomic potential, and copy number. In otherexamples, a transfer cassette with the appropriate recombination site(s)is introduced into the cell or the organism having the target sitedescribed above. A recombinase that recognizes and implementsrecombination at the recombination sites is provided.

In another example a plurality of copies of the polynucleotide ofinterest is provided to the organism, such as a plant. In some examplesthis is accomplished by the incorporation of an extrachromosomalreplicon into the transfer cassette (see WO 99/25855). In specificexamples, the transfer cassette comprises a replicon and apolynucleotide of interest flanked by a directly repeated first andsecond recombination site, wherein the recombination sites arerecombinogenic with respect to one another. When an appropriaterecombinase is provided, the transfer cassette flanked by the directlyrepeated first and second recombination sites is excised from the genomeof the organism, for example a plant, producing a viable repliconcontaining the polynucleotide of interest. Replication of this repliconresults in a high number of copies of the replicon, the polynucleotideof interest, and/or prolongs the availability of the transfer cassettewithin the cell. In other examples, a third functional recombinationsite is present between the replicon and the polynucleotide of interest,wherein the third and the first recombination sites are functional sitesand dissimilar and non-recombinogenic with respect to one another, andthe presence of the appropriate recombinase allows integration of thepolynucleotide of interest into a target site flanked by the third andthe first recombination sites. In one example, at least one of therecombination sites used in the method comprises a functional, modifiedFRT recombination site provided herein.

A replicon comprises an extrachromosomal self-replicating unit. Thereplicon can originate from a virus, plasmid or cell and has thecapacity for self-replication. In this example, the transfer cassettecomprises both a replicon and the polynucleotide of interest. In oneexample, an organism having a target site stably incorporated into itsgenome is provided. A transfer cassette comprising in a 5′ to 3′ or 3′to 5′ orientation: a first functional recombination site, a replicon, asecond functional recombination site, the polynucleotide of interest,and a third functional recombination site is provided. The first andthird recombination site of this transfer cassette are directlyrepeated, corresponding and recombinogenic with respect to each, and thesecond recombination site is dissimilar and non-recombinogenic withrespect to the first and the third recombination sites, wherein at leastone of the first, the second, or the third recombination sites comprisea functional modified FRT recombination site comprising a spacersequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. The transfercassette can be contained in a T-DNA. In one example, the replicon is aviral replicon. A viral replicon is any DNA or RNA derived from a virusthat undergoes episomal replication in a host cell. It containscis-acting viral sequences necessary for replication, for example thereplication origin. It may or may not contain trans-acting viralsequences needed for replication. The excised viral DNA is capable ofacting as a replicon or replication intermediate, either independently,or with factors supplied in trans. The viral DNA may or may not encodeinfectious viral particles and furthermore may contain insertions,deletions, substitutions, rearrangements or other modifications. Theviral DNA may contain heterologous DNA. In this case, heterologous DNArefers to any non-viral DNA or DNA from a different virus. For example,the heterologous DNA may comprise an expression cassette for a proteinor RNA of interest.

Viral replicons suitable for use in the methods and compositions includethose from geminivirus, begomovirus, curtovirus, or mastrevirus,(−)strand RNA viruses, (+) strand RNA viruses, potyvirus, potexvirus,and tobamovirus. Viral replicons can also include those of viruseshaving a circular DNA genome or replication intermediate, such as:Abuitilon mosaic virus (AbMV), African cassava mosaic virus (ACMV),banana streak virus (BSV), bean dwarf mosaic (BDMV), bean golden mosaicvirus (BGMV), beet curly top virus (BCTV), beet western yellow virus(BWYV) and other luteoviruses, cassava latent virus (CLV), camationetched virus (CERV), cauliflower mosaic virus (CaMV), chloris striatemosaic virus (CSMV), commelina yellow mottle virus (CoYMV), cucumbermosaic virus (CMV), dahlia mosaic virus (DaMV), digitaria streak virus(DSV), figwort mosaic virus (FMV), hop stunt viroid (HSV), maize streakvirus (MSV), mirabilias mosaic virus (MMV), miscanthus streak virus(MiSV), potato stunt tuber virus (PSTV), panicum streak virus (PSV),potato yellow mosaic virus (PYMV), potato virus X (PVX), rice tungrobacilliform virus (RTBV), soybean chlorotic mottle virus (SoyCMV),squash leaf curl virus (SqLCV), strawberry vein banding virus (SVBV),sugarcane streak virus (SSV), thistle mottle virus (ThMV), tobaccomosaic virus (TMV), tomato golden mosaic virus (TGMV), tomato mottlevirus (TMoV), tobacco ringspot virus (TobRV), tobacco yellow dwarf virus(TobYDV), tomato leaf curl virus (TLCV), tomato yellow leaf curl virus(TYLCV), tomato yellow leaf cudr virus-Thailand (TYLCV-t) and wheatdwarf virus (WDV) and derivatives thereof. In some examples, the viralreplicon may be from ACMV, MSV, WDV, TGMV or TMV.

In other examples, the insertion of a polynucleotide of interest intothe genome of the organism occurs via a single cross over event. Forinstance, the transfer cassette can comprise a first recombination site,a replicon, a polynucleotide of interest, and a second recombinationsite. The first and second recombination sites of the transfer cassetteare recombinogenic, dissimilar or corresponding, and directly repeatedwith respect to one another. The target site can comprise a singlerecombination site that is recombinogenic to one of the recombinationsites of the transfer cassette. Such recombinogenic recombination sitescan be designed such that integrative recombination events are favoredover the excision reaction. Such recombinogenic recombination sites areknown, for example, Albert et al. introduced nucleotide changes into theleft 13 bp element (LE mutant lox site) or the right 13 bp element (REmutant lox site) of the lox site. Recombination between the LE mutantlox site and the RE mutant lox site produces the wild-type loxP site anda LE+RE mutant site that is poorly recognized by the recombinase Cre,resulting in a stable integration event (Albert et al. (1995) Plant J7:649-659). See also, for example, Araki et al. (1997) Nucleic Acids Res25:868-872.

The transfer cassette is introduced into the organism comprising thetarget site. When an appropriate recombinase is provided, arecombination event between the recombinogenic recombination sites ofthe transfer cassette occurs. This event results in excision of thereplicon, which may assume a circularized form. Replication of thereplicon unit results in a high copy number of the replicon in theorganism and prolongs the availability of the transfer cassette in thecell. A second recombination event between the recombinogenicrecombination sites of the target site and transfer cassette allows thestable integration of the replicon unit and the polynucleotide ofinterest at the target site of the organism.

The methods provide for the targeted insertion of a polynucleotide ofinterest. If the polynucleotide of interest is introduced into anorganism, it may impart various changes in the organism, particularlyplants, including, but not limited to, modification of the fatty acidcomposition in the plant, altering the amino acid content of the plant,altering pathogen resistance, and the like. These results can beachieved by providing expression of heterologous products, increasedexpression of endogenous products in plants, or suppressed expression ofendogenous produces in plants.

General categories of polynucleotides of interest include for example,those genes involved in information, such as zinc fingers, thoseinvolved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes include for example, sequences encoding traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, oil, starch, carbohydrate, phytate,protein, nutrient, metabolism, digestability, kernel size, sucroseloading, and commercial products. Traits such as oil, starch, andprotein content can be genetically altered. Modifications includeincreasing content of oleic acid, saturated and unsaturated oils,increasing levels of lysine and sulfur, providing essential amino acids,and also modification of starch. Hordothionin protein modifications toalter amino acid levels are described in U.S. Pat. Nos. 5,703,049,5,885,801, 5,885,802, 5,990,389. Other examples are a lysine and/orsulfur rich seed protein encoded by the soybean 2S albumin described inU.S. Pat. No. 5,850,016, and the chymotrypsin inhibitor from barley,described in Williamson et al. (1987) Eur J Biochem 165:99-106.

Derivatives of the coding sequences can be made to increase the level ofpreselected amino acids in the encoded polypeptide. For example,polynucleotides encoding the barley high lysine polypeptide (BHL) arederived from barley chymotrypsin inhibitor (WO 98/20133). Other proteinsinclude methionine-rich plant proteins such as from sunflower seed(Lilley et al. (1989) Proceedings of the World Congress on VegetableProtein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite(American Oil Chemists Society, Champaign, Ill.), pp. 497-502); corn(Pedersen et al. (1986) J Biol Chem 261:6279; Kirihara et al. (1988)Gene 71:3); and rice (Musumura et al. (1989) Plant Mol Biol 12:123).

Insect resistance polynucleotides may encode resistance to pests such asrootworm, cutworm, European Corn Borer, and the like. Suchpolynucleotides include, for example, Bacillus thuringiensis toxicprotein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514;5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); and thelike.

Polynucleotides encoding disease resistance traits includedetoxification genes, such as against fumonosin (U.S. Pat. No.5,792,931); avirulence (avr) and disease resistance (R) genes (Jones etal. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; andMindrinos et al. (1994) Cell 78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides such aschlorosulfuron (e.g., the S4 and/or Hra mutations in ALS); genes codingfor resistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene);glyphosate (e.g., the EPSPS gene or the GAT gene; see for example patentpublications US20040082770 and WO 03/092360) or other known genes.Antibiotic resistance can also be provided, for example the nptII geneencodes resistance to the antibiotics kanamycin and geneticin.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

Commercial traits can also be encoded on a gene or genes that could, forexample increase starch for ethanol production, or provide expression ofproteins. Another commercial use of transformed plants is the productionof polymers and bioplastics such as described in U.S. Pat. No.5,602,321. Genes such as β-Ketothiolase, PHBase (polyhydroxyburyratesynthase), and acetoacetyl-CoA reductase (see Schubert et al. (1988) JBacteriol 170:5837-5847) facilitate expression of polyhydroxyalkanoates(PHAs).

Reduction of the activity of specific genes (also known as genesilencing, or gene suppression) is desirable for several aspects ofgenetic engineering in plants. Many techniques for gene silencing arewell known, including but not limited to antisense technology (see,e.g., Sheehy et al. (1988) Proc Natl Acad Sci USA 85:8805-8809; and U.S.Pat. Nos. 5,107,065; 5,453,566; and 5,759,829); cosuppression (e.g.,Taylor (1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech8:340-344; Flavell (1994) Proc Natl Acad Sci USA 91:3490-3496; Finneganet al. (1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) MolGen Genet 244:230-241); RNA interference (Napoli et al. (1990) PlantCell 2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev13:139-141; Zamore et al. (2000) Cell 101:25-33; Javier (2003) Nature425:257-263; and, Montgomery et al. (1998) Proc Natl Acad Sci USA95:15502-15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691-705; and Baulcombe (1999) Curr Op Plant Bio2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334:; 585-591); hairpin structures (Smith et al. (2000) Nature407:319-320; WO 99/53050; WO 02/00904; and WO 98/53083); ribozymes(Steinecke et al. (1992) EMBO J 11:1525; U.S. Pat. No. 4,987,071; and,Perriman et al. (1993) Antisense Res Dev 3:253); oligonucleotidemediated targeted modification (e.g., WO 03/076574 and WO 99/25853);Zn-finger targeted molecules (e.g., WO 01/52620; WO 03/048345; and WO00/42219); and other methods or combinations of the above methods known.

The polynucleotides can be provided in a DNA construct. In addition, inspecific examples recombination sites and/or the polynucleotide encodingan appropriate recombinase is also contained in the DNA construct. Thecassette can include 5′ and 3′ regulatory sequences operably linked tothe polynucleotide of interest. Alternatively, the DNA construct flankedby the appropriate recombination site can lack the 5′ and/or 3′regulatory elements. In this instance, the DNA construct is designedsuch that in the presence of the appropriate recombinase a recombinationevent at the target site will result in the 5′ and/or 3′ regulatoryregions being operably linked to the sequences of the DNA construct.Intervening sequences can be present between operably linked elementsand not disrupt the functional linkage. The cassette may additionallycontain at least one additional gene to be introduced into the organism.Alternatively, the additional gene(s) can be provided on multiple DNAconstructs. Such a DNA construct may be provided with a plurality ofrestriction sites or recombination sites for insertion of the sequenceof interest to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectableand/or screenable marker genes.

In some examples, the DNA construct can include in the 5′ to 3′direction of transcription, a transcriptional and translationalinitiation region, a polynucleotide of interest, and a transcriptionaland translational termination region functional in the organism ofinterest. In other examples, the DNA construct comprises apolynucleotide of interest 3′ to a recombination site. In this example,the target site can comprise a promoter 5′ to the correspondingrecombination site, thereby, upon recombination, the nucleotide sequenceof interest is operably linked to the promoter sequence. The variousrecombination sites provided herein can be positioned anywhere in theDNA construct, including the 5′ UTR, 3′ UTR, regulatory regions, intronsand/or coding sequence.

The transcriptional initiation region, the promoter, may be native,analogous, foreign, or heterologous to the host organism or to thepolynucleotide of interest. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. Such constructsmay change expression levels of the polynucleotide of interest in theorganism. The termination region may be native or heterologous with thetranscriptional initiation region, it may be native or heterologous withthe operably linked polynucleotide of interest, or it may be native orheterologous with the host organism. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also Guerineauet al. (1991) Mol Gen Genet 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res 17:7891-7903; and Joshi et al.(1987) Nucleic Acids Res 15:9627-9639. The nucleotide sequence ofinterest can also be native or analogous or foreign or heterologous tothe host organism,

Where appropriate, the codon usage in the nucleotide sequence ofinterest or the recombinase may be modified for expression in thetransformed organism. For example, the genes can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell & Gowri (1990) Plant Physiol 92:1-11 for a discussion ofhost-preferred codon usage. Methods are available for synthesizingplant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and5,436,391, WO 99/25841, and Murray et al. (1989) Nucleic Acids Res17:477-498.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The DNA construct may additionally contain 5′ leader sequences. Suchleader sequences can act to enhance translation. Translation leaders areknown and include picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc Natl Acad Sci USA 86:6126-6130); potyvirus leaders, for example,TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165:233-238),MDMV leader (Maize Dwarf Mosaic Virus) (Allison et al. (1986) Virology154:9-20; and Kong et al. (1988) Arch Virol 143:1791-1799), and humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) inMolecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol84:965-968. Other methods or sequences known to enhance translation canalso be utilized, for example, introns, and the like.

In preparing the DNA construct, the various DNA fragments may bemanipulated to place the sequences in the proper orientation and, asappropriate, in the proper reading frame. Toward this end, adapters orlinkers may be employed to join the DNA fragments or other manipulationsmay be involved to provide for convenient restriction sites, removal ofsuperfluous DNA, removal of restriction sites, or the like. For thispurpose, in vitro mutagenesis, primer repair, restriction, annealing,resubstitutions, transitions and/or transversions, may be involved.

Generally, the DNA construct will comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues and have beendiscussed in detail elsewhere herein, as well as, exemplary promoters ofinterest.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1. Generating Libraries Comprising Modified FRTRecombination Sites

Two complementary degenerate oligos containing FRT sequences with the 6central spacer positions being randomly mutagenized were synthesized atSynthetic Genetics (San Diego, Calif.): Oligo1:5′-gccagcatgcaagcttgaattccgaagttcctatactNNNNNNagaataggaacttcgagatctggatccgcggaacg-3′ (SEQ ID NO:52); and Oligo2:5′-cgttccgcggatccagatctcgaagttcctattctNNNNNNagtataggaacttcggaattcaagcttgcatgctggc-3′ (SEQ ID NO:53).

The spacer region is 8 bp. In this experiment, the central 6 bp regionwas targeted for modification, hence, the other two nucleotides are keptunchanged. One pmol of oligo 1 and one pmol of oligo 2 were annealed byheat denaturation at 95° C. for 2 minutes followed by gradual cooling toroom temperature. Annealed oligos were digested with EcoRI and BamHI andligated into the EcoRI/BamHI sites of a pSportI-derived vector to which3 additional bases created a HpaI restriction site (BRL LifeTechnologies, Gaithersburg, Md.) and the PHP13273 vector containing aspectinomycin resistance gene to create a two modified FRT plasmidlibraries. A 10:1 and 4:1 molar ratio of annealed oligo to pSport andPHP13273 was used for the respective ligation reactions. Under theseligation conditions, 10 out of 10 randomly picked colonies were found tocontain a monomeric insertion of a modified FRT site. The modified FRTlibrary referred to as “library A” is in the pSport vector and carriesthe antibiotic resistant marker ampicillin. The modified FRT library,referred to as “library B” is in the PHP13273 vector and carries theantibiotic resistant marker spectinomycin. A total number of 15,904colonies were collected to make FRT library A and 19,600 colonies werecollected to make the FRT library B. This represented a 4× coverage ofthe central 6 positions (4⁸=4096) in the FRT site. Plasmid DNA wasisolated from these two libraries and used for library scale screening.

Example 2. Library Scale Screening to Identify Recombinogenic ModifiedFRT Recombination Sites

An equal molar amount of DNA from each of the modified FRT libraries Aand B were mixed into one tube containing reaction buffer forFLP-mediated in vitro recombination. A typical 20 μl recombinationreaction comprises 25 mM Tris Cl at pH 7.5, 10 mM MgCl₂, 5 mM DTT, 50fmol library A DNA, 50 fmol library B DNA, and 2 μl FLP (0.07 μg/μlfinal). The reaction is carried out at 30° C., and aliquots taken atvarious time points. At each time point, 2 μl aliquots were taken andthe reaction stopped by boiling for 1 min with gradual cooling to roomtemperature. Typically, samples were taken at 0, 2, 5, 10, 30, 60, and90 minutes and could be used to evaluate fast vs slow reactive FRTsites. If only one time point was taken, the 90 minute timepoint wasused.

Reaction samples were transferred into E. coli DH5α cells according tostandard procedures. Equal aliquots of each transformation mixture wasspread onto one plate each containing either ampicillin only,spectinomycin only, or containing both ampicillin and spectinomycin.Successfully recombined co-integrant DNA will carry both selectionmarkers and thus, after transfer into E. coli, will confer resistance toboth ampicillin and spectinomycin.

Those colonies with resistance to both antibiotics were picked andplasmid DNA was prepared using Montage 96-well HTP plasmid DNApreparation kit (Millipore, Billerica, Mass. USA). Candidate FRT siteswere obtained by PCR using primers flanking recombined FRT sites in theco-integrate DNA. The PCR primers used were Forward primer:5′-gcacatacaaatggacgaacgga-3 (SEQ ID NO:54) and Reverse primer:5′-cctcttcgctattacgccagct-3′(SEQ ID NO:55). The PCR conditions were asfollows: One cycle: 95° C., 1 min; 20 cycles: 95° C., 30 sec; 61° C., 2min; one cycle: 67° C., 3 min; Hold: 4° C. The sequence of the amplifiedcandidate FRT sites was determined by Cycle sequencing (essentially asdescribed in Slatko et al. (1993) DNA Sequencing. In, Current Protocolsin Molecular Biology, (ed. By Ausubel et al.) Ch. 7, pp 7.0.1-7.6.13.New York: John Wiley & Sons).

Example 3. Methods for Assaying Excision Efficiency of RecombinogenicModified FRT Recombination Sites

To assay recombinase-mediated excision efficiency of a candidate FRTsite, excision vectors were made in which two copies of a candidaterecombinogenic modified FRT site were cloned in direct orientationflanking the maize ubiquitin promoter sequence in pSport (BRL LifeTechnologies, Gaithersburg, Md.). An excision reaction was carried outunder the following conditions: 3 μl miniprep excision vector DNA (2mg/ml), 1 μl 10× buffer (250 mM Tris-CI at pH 7.5, 100 mM MgCl₂, 50 mMDTT), 5 μl ddH₂O, and 1 μl FLP (0.72 mg/ml). The reaction mixture wasincubated at 30° C. for 30 min, boiled for 2 min, cooled to roomtemperature, digested with EcoRV and XhoI, and then subjected to agarosegel electrophoresis.

EcoRV generates a single cut in the pSport vector backbone while XhoIgenerates a single cut in the sequence of maize ubiquitin promoter.Double digestion of the non-recombined excision vector produces twofragments of 4332 bp and 769 bp. Double digestion of the product vectorafter excision takes place produces an additional fragment of 952 bp.The DNA fragments were quantified using Quantity One software fromBio-Rad Laboratories. As excision occurs, an increased amount of the 952bp fragment is produced and less of the 769 bp fragment is produced.Thus, the ratio of the 952 bp fragment to that of the 769 bp fragmentmeasures absolute excision efficiency. In this experiment, relativerecombination excision efficiency (% excision efficiency) of a FRT siteis calculated as the excision efficiency in the presence of native yeastFLP of a first modified FRT site with a second modified FRT site dividedby the excision efficiency of a pair of wild-type FRT site (SEQ IDNO:39)×100%.

Various modified FRT recombination sites identified in the methods ofExample 2 were analyzed for their ability to retain biological activity.Table 1 sets forth various functional modified FRT recombination sitesand their relative recombination efficiency determined as outlinedabove.

TABLE 1 SEQ ID NO for SEQ ID No minimal for modi- Excision FRT modifiedSpacer fied spacer Efficiency sites FRT site Sequence sequence (%) FRT139 TTTCTAGA 43 100 FRT12 21 TCTATGTA  1 102 FRT57 22 TTTTCTAA  2  82FRT85 23 TTTCTTGA  3 116 FRT87 24 TTTCTGGA  4  93 FRT53 25 TGTAAAAA  5 64 FRT62 26 TTTAGGTA  6  72 FRT78 27 TGAAAAGA  7  60 FRT34 28 TGTAATGA 8  34 FRT70 29 TATACAAA  9  25 FRT78 30 TTCCATAA 10  30 FRT89 31TCTCTAGA 11  39 FRT43 32 TTCCGAGA 12  14 FRT45 33 TCTCTTGA 13   5 FRT5534 TCCACAGA 14   7 FRT65 35 TGATTGGA 15  18 FRT69 36 TTTTGTGA 16  9FRT74 37 TGAGAGAA 17   5 FRT86 38 TTTCTCGA 18  12 FRT5 40 CTTTTGAA 44 15 *The spacer sequences were flanked by the wild type symmetrical 13base pair element set forth in FIG. 1.

Example 4. Methods for Assaying Co-Integration Efficiency ofRecombinogenic Modified FRT Recombination Sites

The experiment was carried out as described in Example 2. Briefly, FRT1,5, and 6 (SEQ ID NOS:39, 40, and 41) were individually cloned intoEcoRI/BamHI sites of PHP13273 and modified pSport1 vector. 50 fmol DNAof FRT1 in PHP13273 (Spec^(r)) was mixed with 50 fmol DNA of FRT1 inmodified pSport1 (Ap^(r)) in 20 μl reaction buffer containing 25 mM TrisCl at pH 7.5, 10 mM MgCl₂, 5 mM DTT, and 2 μl FLP (0.07 μg/μl final). Ateach time point, 2 μl aliquots were taken and the reaction stopped byboiling for 1 min with gradual cooling to room temperature. Reactionsamples were transferred into E. coli DH5α cells according to standardprocedures. Equal aliquots of each transformation mixture were spreadonto one plate each containing ampicillin only, spectinomycin only, orcontaining both ampicillin and spectinomycin. Successfully recombinedco-integrate DNA via FRT1 sites will carry both selection markers andthus, after transfer into E. coli, will confer resistance to bothampicillin and spectinomycin. Those colonies with resistance to bothantibiotics were picked and co-integrated plasmid DNA was prepared forfurther analysis. Clones that are resistant to both antibiotics but donot harbor co-integrated plasmid DNA were subtracted in the calculationof co-integration frequency.

Co-integration frequency of FRT1 was determined by calculating thepercentage of colonies harboring co-integrated plasmid DNA amongcolonies resistant to one antibiotic drug. Similarly, in vitrointegration of FRT5 or FRT6 was performed and co-integration frequencyof FRT5 or FRT6 was determined accordingly. The results are shown inTable 2.

TABLE 2 Percentage of co-integrants recovered from in vitro FLP-mediatedrecombination (%) Time (h) 0 0.5 1.0 1.5 2.0 FRT1 + FRT1 0.01 0.32 0.700.98 1.03 FRT5 + FRT5 0.00 0.04 0.04 0.08 0.09 FRT6 + FRT6 0.02 0.200.18 0.28 0.27

FLP-mediated in vitro recombination was performed as described before.When DNA containing dissimilar FRT sites are mixed in the reaction, suchas in the previously described library-scale screening, intermolecularrecombination between two corresponding FRT sites is further reduced. Inreactions containing FRT1 sites only, FRT5 sites only, or FRT6 sitesonly, recombination between FRT1 sites is approximately 10-fold moreefficient than between FRT5 sites and approximately 4-fold moreefficient than between FRT6 sites (Table 2).

In this example, plasmid DNA containing three different FRT sites (FRT1.FRT5, and FRT6), each residing on modified pSport1 carrying Ap^(r)selectable marker and PHP13273 carrying Spec^(r.) were mixed in thereaction. Among FRT1, FRT5, and FRT6, two different FRT sites do notrecombine with each other. In the reaction having equimolar amount ofDNA containing FRT sites of FRT1, FRT5, and FRT6, recombinationefficiency between any two corresponding FRT sites is reduced. Theresults are shown in Table 3. The combined co-integration frequencybetween two FRT1 sites, two FRT5 sites, and two FRT6 sites was 0.09%after 90 minutes, approximately 10-fold less than that of the reactionhaving the FRT1 site only. The majority of co-integrates is fromrecombination via the most efficient FRT sites, FRT1, in the reaction asindicated by the fact that all of the 10 randomly picked co-integrateswere recombination products of FRT1 sites. In the reaction having lowermolar amount of DNA containing FRT1 site (molar ratio among FRT1, FRT5,and FRT6 is: 0.04:1.00:1.00), the overall recombination was furtherreduced. Furthermore, none of the 10 randomly picked co-integrates wasrecombination product of FRT1 sites.

TABLE 3 FRT1 co-integrate/ Co- Total integrate co-integrate FRT Sites*(%) analysed FRT1 (Ap^(r), 50 fmol) + FRT1 (Spec^(r), 50 fmol) 0.9810/10 FRT1 (Ap^(r), 50 fmol) + FRT5 (Spec^(r), 50 fmol) 0.08 NA FRT1(Ap^(r), 50 fmol) + FRT6 (Spec^(r), 50 fmol) 0.28 NA FRT1 (Ap^(r), 16fmol) + FRT1 (Spec^(r), 16 0.09 10/10 fmol) + FRT5 (Ap^(r), 16 fmol) +FRT5 (Spec^(r), 16 fmol) + FRT6 (Ap^(r), 16 fmol) + FRT6 (Spec^(r), 16fmol) FRT1 (Ap^(r), 0.8 fmol) + FRT1 (Spec^(r), 0.07  0/10 0.8 fmol) +FRT5 (Ap^(r), 20 fmol) + FRT5 (Spec^(r), 20 fmol) + FRT6 (Ap^(r), 20fmol) + FRT6 (Spec^(r), 20 fmol) *Selection marker and molar amount ofDNA used in the reaction are included in parenthesis.

Example 5: Plant Transformation A. Particle Bombardment Transformationand Regeneration of Maize Callus

Immature maize embryos from greenhouse or field grown High type II(HiII) donor plants are bombarded with an isolated polynucleotidecomprising a recombination site, transfer cassette, target site, and/orrecombinase provided herein. If the polynucleotide does not include aselectable marker, another polynucleotide containing a selectable markergene can be co-precipitated on the particles used for bombardment. Forexample, a plasmid containing the PAT gene (Wohlleben et al. (1988) Gene70:25-37) which confers resistance to the herbicide Bialaphos can beused. Transformation is performed as follows.

The ears are surface sterilized in 50% Chlorox bleach plus 0.5% Microdetergent for 20 minutes, and rinsed two times with sterile water. Theimmature embryos are excised and placed embryo axis side down (scutellumside up), 25 embryos per plate. These are cultured on 560L agar medium 4days prior to bombardment in the dark. Medium 560L is an N6-based mediumcontaining Eriksson's vitamins, thiamine, sucrose, 2,4-D, and silvernitrate. The day of bombardment, the embryos are transferred to 560Ymedium for 4 hours and are arranged within the 2.5-cm target zone.Medium 560Y is a high osmoticum medium (560L with high sucroseconcentration).

A plasmid vector comprising a polynucleotide of interest operably linkedto the selected promoter is constructed. This plasmid DNA, plus plasmidDNA containing a PAT selectable marker if needed, is precipitated onto1.0 μm (average diameter) gold pellets using a CaCl2 precipitationprocedure as follows: 100 μl prepared gold particles (0.6 mg) in water,20 μl (2 μg) DNA in TrisEDTA buffer (1 μg total), 100 μl 2.5 M CaCl2, 40μl 0.1 M spermidine.

Each reagent is added sequentially to the gold particle suspension. Thefinal mixture is sonicated briefly. After the precipitation period, thetubes are centrifuged briefly, liquid removed, washed with 500 μl 100%ethanol, and centrifuged again for 30 seconds. Again the liquid isremoved, and 60 μl 100% ethanol is added to the final gold particlepellet. For particle gun bombardment, the gold/DNA particles are brieflysonicated and 5 μl spotted onto the center of each macrocarrier andallowed to dry about 2 minutes before bombardment.

The sample plates are bombarded at a distance of 8 cm from the stoppingscreen to the tissue, using a DuPont biolistics helium particle gun. Allsamples receive a single shot at 650 PSI, with a total of ten aliquotstaken from each tube of prepared particles/DNA.

Four to 12 hours post bombardment, the embryos are moved to 560P (a lowosmoticum callus initiation medium similar to 560L but with lower silvernitrate), for 3-7 days, then transferred to 560R selection medium, an N6based medium similar to 560P containing 3 mg/liter Bialaphos, andsubcultured every 2 weeks. After approximately 10 weeks of selection,callus clones are sampled for PCR and/or activity of the polynucleotideof interest. Positive lines are transferred to 288J medium, an MS-basedmedium with lower sucrose and hormone levels, to initiate plantregeneration. Following somatic embryo maturation (2-4 weeks),well-developed somatic embryos are transferred to medium for germinationand transferred to the lighted culture room. Approximately 7-10 dayslater, developing plantlets are transferred to medium in tubes for 7-10days until plantlets are well established. Plants are then transferredto inserts in flats (equivalent to 2.5″ pot) containing potting soil andgrown for 1 week in a growth chamber, subsequently grown an additional1-2 weeks in the greenhouse, then transferred to Classic™ 600 pots (1.6gallon) and grown to maturity. Plants are monitored for expression ofthe polynucleotide of interest.

B. Agrobacterium-Mediated Transformation and Regeneration of MaizeCallus

For Agrobacterium-mediated transformation of maize, a polynucleotidecomprising a recombination site, transfer cassette, target site, and/orrecombinase provided herein is used with the method of Zhao (U.S. Pat.No. 5,981,840).

Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium containing a polynucleotideof interest, where the bacteria are capable of transferring thenucleotide sequence of interest to at least one cell of at least one ofthe immature embryos (step 1: the infection step). In this step theimmature embryos are immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). Following thisco-cultivation period an optional “resting” step may be performed (step3: resting step). The immature embryos are cultured on solid medium withantibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos are cultured on medium containing a selective agentand growing transformed callus is recovered (step 4: the selectionstep). The immature embryos are cultured on solid medium with aselective agent resulting in the selective growth of transformed cells.The callus is then regenerated into plants (step 5: the regenerationstep), and calli grown on selective medium are cultured on solid mediumto regenerate the plants.

C. Transformation of Dicots

A polynucleotide comprising a recombination site, transfer cassette,target site, and/or recombinase provided herein can be introduced intoembryogenic suspension cultures of soybean by particle bombardment usingessentially the methods described in Parrott, et al. (1989) Plant CellRep. 7:615-617. This method, with modifications, is described below.

Seed is removed from pods when the cotyledons are between 3 and 5 mm inlength. The seeds are sterilized in a bleach solution (0.5%) for 15minutes after which time the seeds are rinsed with sterile distilledwater. The immature cotyledons are excised by first cutting away theportion of the seed that contains the embryo axis. The cotyledons arethen removed from the seed coat by gently pushing the distal end of theseed with the blunt end of the scalpel blade. The cotyledons are thenplaced in Petri dishes (flat side up) with SB1 initiation medium (MSsalts, B5 vitamins, 20 mg/L 2,4-D, 31.5 g/L sucrose, 8 g/L TC Agar, pH5.8). The Petri plates are incubated in the light (16 hr day; 75-80 μE)at 26° C. After 4 weeks of incubation the cotyledons are transferred tofresh SB1 medium. After an additional two weeks, globular stage somaticembryos that exhibit proliferative areas are excised and transferred toFN Lite liquid medium (Samoylov, et al. (1998) In Vitro Cell Dev.Biol.—Plant 34:8-13). About 10 to 12 small dusters of somatic embryosare placed in 250 ml flasks containing 35 ml of SB172 medium. Thesoybean embryogenic suspension cultures are maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights (20μE) on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures are then transformed usingparticle gun bombardment (Klein et al. (1987) Nature 327:70; U.S. Pat.No. 4,945,050). A BioRad Biolisticä PDS1000/HE instrument can be usedfor these transformations. A selectable marker gene, which is used tofacilitate soybean transformation, is a chimeric gene composed of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225(from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3′ region ofthe nopaline synthase gene from the T-DNA of the Ti plasmid ofAgrobacterium tumefaciens.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl2(2.5 M). The particle preparation is agitated for three minutes, spun ina microfuge for 10 seconds and the supernatant removed. The DNA-coatedparticles are washed once in 400 μL 70% ethanol and resuspended in 40 μLof anhydrous ethanol. The DNA/particle suspension is sonicated threetimes for one second each. Five μL of the DNA-coated gold particles arethen loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. Membrane rupture pressure is set at 1100 psi andthe chamber is evacuated to a vacuum of 28 inches mercury. The tissue isplaced approximately 8 cm away from the retaining screen, and isbombarded three times. Following bombardment, the tissue is divided inhalf and placed back into 35 ml of FN Lite medium.

Five to seven days after bombardment, the liquid medium is exchangedwith fresh medium. Eleven days post bombardment the medium is exchangedwith fresh medium containing 50 mg/mL hygromycin. This selective mediumis refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue will be observed growing from untransformed, necroticembryogenic dusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line is treated as anindependent transformation event. These suspensions are then subculturedand maintained as clusters of immature embryos, or tissue is regeneratedinto whole plants by maturation and germination of individual embryos.

D. DNA Isolation from Callus and Leaf Tissues

Putative transformation events can be screened for the presence of thetransgene. Genomic DNA is extracted from calli or leaves using amodification of the CTAB (cetyltriethylammonium bromide, Sigma H5882)method described by Stacey and Isaac (1994 In Methods in MolecularBiology Vol. 28, pp. 9-15, Ed. P. G. Isaac, Humana Press, Totowa, N.J.).Approximately 100-200 mg of frozen tissue is ground into powder inliquid nitrogen and homogenised in 1 ml of CTAB extraction buffer (2%CTAB, 0.02 M EDTA, 0.1 M Tris-CI pH 8, 1.4 M NaCl, 25 mM DTT) for 30 minat 65° C. Homogenised samples are allowed to cool at room temperaturefor 15 min before a single protein extraction with approximately 1 ml24:1 v/v chloroform:octanol is done. Samples are centrifuged for 7 minat 13,000 rpm and the upper layer of supernatant collected usingwide-mouthed pipette tips. DNA is precipitated from the supernatant byincubation in 95% ethanol on ice for 1 h. DNA threads are spooled onto aglass hook, washed in 75% ethanol containing 0.2 M sodium acetate for 10min, air-dried for 5 min and resuspended in TE buffer. Five μl RNAse Ais added to the samples and incubated at 37° C. for 1 h. Forquantification of genomic DNA, gel electrophoresis is performed using a0.8% agarose gel in 1×TBE buffer. One microlitre of each of the samplesis fractionated alongside 200, 400, 600 and 800 ng μl-1 λ uncut DNAmarkers.

Example 6. Comparing Relative Recombination Efficiency of Dissimilar FRTSequences in Maize Cells

Two assays are provided that measure relative transgene activation ratesas a result of FLP-mediated excision, which brings a promoter andtransgene into functional proximity. The method can be used tocharacterize the recombination efficiency of either corresponding and/ordissimilar recombination sites and thereby determine if the sites arerecombinogenic or non-recombinogenic with one another.

Two assays are discussed below: (A) scoring activation of YellowFluorescence Protein (YFP) in individual cells and (B) scoringluciferase activity.

A. Fluorescence Assay

Three transgenic expression cassettes (outlined in Table 4) areintroduced in either a FRT-Test treatment or a control treatment.

TABLE 4 FRT-Test construct Control construct CPN60:FRTx:GUS:FRTx:YFP:35sterm CPN60:FRTx:YFP:35s term Actin::CFP::nos Actin::CFP::nosUbi::FLP::pniII Ubi::FLP::pniII YFP = yellow fluorescence protein; CFP =cyan fluorescence protein; CPN60 = maize chaparonin 60 promoter (Close(1993) Master's Thesis, Iowa State University).

In both the control and FRT-testing treatments, the relevant (orappropriate) three expression cassettes are mixed in equimolar ratios,and introduced into scutellar cells of Hi-II immature embryos usingstandard particle delivery methods. After two days, the numbers of cyan-and yellow-fluorescent cells are counted using a Leica epifluorescentstereomicroscope. The number of cyan-fluorescing cells is used tonormalize between treatments by providing a relative measure of how manycells received sufficient DNA to express the transgenes. To validatethis assay system, FRT1 is used for the first experiment. As a controltreatment, a mixture of the following three plasmids is used:Actin::Cyan FP::nos, CPN60:FRT1:YFP:35s term, and Ubi::FLP::pinII. Inthe control treatment, when these three plasmids are co-introduced andthe numbers of cyan and yellow cells are scored two days later, thenumbers of cyan and yellow cells in the population is expected to beapproximately equivalent (1:1).

In the FRT-Test treatment, when FRT1 is used in the excision-activatedcassette (CPN60:FRT1:GUS:FRT1:YFP:35s term), it is expected thatapproximately 90-95% of the cells expressing cyan fluorescence alsoexpress yellow fluorescence, i.e. excision of the FRT1-flanked region isrelatively efficient. Based on previous studies with FRT5, when FRT5 isused in the excision cassette, the frequency of cyan fluorescing cellsthat also express the yellow fluorescent protein is expected to drop toapproximately 15% of that observed in the FRT1 treatment.

The excision-activated cassette can also be used to determine if twodissimilar FRT recombination sites are recombinogenic ornon-recombinogenic. To determine if FRT1 and FRT5 are recombinogenic ornon-recombinogenic with respect to one another, an excision-activatedcassette comprising CPN60:FRT1:GUS:FRT5:YFP:35s term is constructed. Asoutlined in Table 4, the three expression cassettes are mixed inequimolar ratios, and introduced into scutellar cells of Hi-II immatureembryos using standard particle delivery methods. After two days, thenumbers of cyan- and yellow-fluorescent cells are counted using a Leicaepifluorescent stereomicroscope. The number of cyan-fluorescing cells isused to normalize between treatments by providing a relative measure ofhow many cells received sufficient DNA to express the transgenes.

When the excision cassette comprises a FRT1 and a FRT5 recombinationsite, the frequency of cyan fluorescing cells that also express theyellow fluorescent protein is expected to drop to approximately lessthan 1% of that observed in when an excision cassette comprising twoFRT1 recombination sites is employed. The sites are therefore determinedto be non-recombinogenic.

B. Assay Based on Activation of Luciferase Enzymatic Activity

The second assay system again uses a mixture of plasmids in equimolaramounts, cobombarded into scutellar cells of Hi-II immature embryos. Forthis assay the three plasmids are shown in Table 5.

TABLE 5 FRT-Test Treatment Control Actin::FRTx::GUS:FRTx:FF-Actin::FRTx:FF-luciferase::nos* luciferase::nos*Nos::Renilla-luciferase::35S term Nos::Renilla-luciferase::35S termUbi::FLP::pniII Ubi::FLP::pniII *FF = firefly luciferase;Renilia-luciferase (Minko et el. (1999) Mol. Gen. Genet. 262: 421-425)

Again, FRT1 is used to validate the assay system. In the controltreatment, Actin:FRT1:FF-luciferase::nos, Nos::Renilla-luciferase::35Sterm, and Ubi::FLP::pinII are introduced into scutellar cells and after2 days the tissue is extracted using methods and solutions provided inthe Promega Dual-luciferase Assay Kit (Promega, Madison, Wis. 53711).Multiple scutella are individually extracted, and the extractssequentially assayed for firefly and then Renilla luciferase activityusing a Fluoroscan. With this mixture of constructs it is expected thatthe expressed firefly luciferase protein produces approximately 5000relative light units and the Renilla luciferase produces about 15,000.When FRT1 is used in the excision-activated cassette(Actin:FRT1:GUS:FRT1:Firefly luci:35s term), the firefly luciferase isexpected to produce about 4500 light units (about 90% of the controltreatment). When FRT 5 is used in the excision cassette, the fireflyluciferase activity is expected to drop to approximately 670 light units(˜15% of FRT1).

Example 7. Targeting the Insertion of a Polynucleotide of Interest intoMaize A. Establishing a Target Line

For evaluation of FRT sequences for site-specific integration, a targetsite is first created by stably integrating a polynucleotide comprisinga target site having two functional FRT recombination sites, where therecombination sites are dissimilar and non-recombinogenic with respectto one another. This initial transformation is accomplished in Hi-IIgermplasm (or inbred lines) using standard Agrobacterium transformationmethods for maize (see Example 5B). For example, to compare the relativeefficiency of FRT5 and FRT87 in the site-specific integration system,the following constructs are separately introduced into Hi-II germplasm:PHP20807:

RB—Ubi:Ubi-intron:FRT1:Yellow Fluorescent Protein::pinII/Ubi:Ubi-intron:GAT::pinII/In2-1 term:GUS:FRT5:Os-Actin-intron:Os-Actin Pro-LB PHP20705:RB—Ubi:Ubi-intron:FRT1:Yellow Fluorescent Protein::pinII/Ubi:Ubi-intron:GAT::pinII/In2-1 term:GUS:FRT87:Os-Actin-intron:Os-Actin Pro-LB

Stable transformants are selected by looking for yellow-fluorescentcallus growing on glyphosate-containing medium. Plants are regeneratedand sent to the greenhouse. Leaf samples are taken for Southernanalysis. Single-copy transgenic plants are grown to maturity andcrossed to wild-type Hi-II (or inbred lines). These transgenic eventsnow contain the FRT1-5 or FRT1-87 target site, and are ready forsite-specific recombinase-mediated recombination.

B. Introduction of the Transfer Cassette Using Particle Bombardment.

Immature embryos from the line having the target sites as evidenced byexpression of yellow fluorescence are used for the subsequentre-transformation. During the re-transformation process, transfercassettes are introduced using standard particle bombardment methods(e.g., see Example 5A). For progeny plants that contain the integratedT-DNA from PHP20705 (the FRT1-FRT87 target site), the following insertcomprising the transfer cassette is used for re-transformation using theparticle gun:

PHP20915:

RB—CaMV35S Term/FRT1:bar::pinII/Ubi:Ubi-intron:Renillaluciferase::pinII/In2-1 term:Am-Cyan1:FRT87/CaMV35S Term-LB.Progeny immature embryos that contain the integrated T-DNA from PHP20807(the FRT1-FRT5 target site) are re-transformed using the particle gunwith the following plasmid:

PHP20954:

RB—CaMV35S Term/FRT1:bar::pinII/Ubi:Ubi-intron:Renillaluciferase::pinII/In2-1 term:Am-Cyan1:FRT5/CaMV35S Term-LB.

For both of the plasmids comprising the transfer cassette (PHP20915 andPHP20954), the bar and Cyan FP genes have no promoter. To reduce thelikelihood that random integration would result in spurious expressionof either gene, the CaMV35S terminator has been placed upstream of theFRT1 site. Each of these plasmids is co-transformed into immatureembryos from their respective target-lines along with plasmid PHP5096(Ubi:Ubi-intron::FLPm::pinII). Either PHP20915 or PHP20954 are mixedwith the FLP-containing plasmid (PHP5096), using 100 ng of theFRT-containing plasmid and 10 ng of the FLP plasmid per bombardment.

To prepare DNA for delivery, DNA solutions are added to 50 μl of agold-particle stock solution (0.1 μg/μl of 0.6 micron gold particles).For example, 10 μl of a 0.1 μg/μl solution of PHP20915 or PHP20954, and10 μl of a 0.01 μg/μl solution of PHP5096 are first added to 30 μl ofwater. To this DNA mixture, 50 μl of the gold stock solution is addedand the mixture briefly sonicated. Next 5 μl of TFX-50 (Promega Corp.,2800 Woods Hollow Road, Madison Wis. 53711) is added and the mixture isplaced on a rotary shaker at 100 rpm for 10 minutes. The mixture isbriefly centrifuged to pellet the gold particles and remove supernatant.After removal of the excess DNA/TFX solution, 120 μl of absolute EtOH isadded, and 10 μl aliquots are dispensed onto the macrocarriers typicallyused with the Dupont PDS-1000 Helium Particle Gun. The gold particleswith adhered DNA are allowed to dry onto the carriers and then these areused for standard particle bombardment. After re-transformation deliveryof the plasmid having the transfer cassette plus the FLP-containingplasmid, the immature embryos are placed onto 560P medium for two weeksto recover, and then moved to medium containing 3 mg/l bialaphos forselection. Successful recombination at both the 5′ (FRT1) and 3′ (FRT87or 5) recombination target sites will result in activation of both thebar gene and the Cyan Fluorescent Protein gene when these structuralgenes are brought into functional proximity with the Ubi or Actinpromoters, respectively. Thus, proper site-specific integration eventswill be selected based on the newly activated phenotypes. When thesecalli are large enough for sampling, genomic DNA is extracted from thetissue, and is analyzed using PCR for the presence of products thatresult from amplification of fragments using primers that span the newlyformed promoter-gene junctions. Finally, leaf samples are taken fromregenerated plants for Southern analysis, to verify proper recombinationto transfer of the donor cassette into the genomic target locus. Oncesuccessful recombinant loci have been verified, plants are grown tomaturity and outcrossed or selfed.

C. Introduction of the Transfer Cassette by Crossing

Transfer cassettes can be provided by sexual crossing. In this examplestable transgenic, single-copy target events are again used containing asingle-copy of the T-DNA cassettes originally delivered fromAgrobacterium containing PHP20705 or PHP20807. However, in this methodstable transgenic donor events are produced using either of two T-DNAAgrobacterium vectors shown below.

1. The donor vector that complements PHP20705: RB-Axig1::LEC1::pinII/UbiPro: Ubi-intron::YFP::pin1/-LB and RB-In2::FLP::pinII-CaMV35STerm/FRT1:bar::pinII/Ubi:Ubi-intron:Renilla luciferase::pinII/In2-1term:Am-Cyan1:FRT87/CaMV35S Term-LB

2. The donor vector that complements PHP20807: RB-Axig1::LEC1::pinII/UbiPro: Ubi-intron::YFP::pinII/-LB and RB-In2::FLP::pinII-CaMV35STerm/FRT1:bar::pinII/Ubi:Ubi-intron:Renilla luciferase::pinII/In2-1term:Am-Cyan1:FRT5/CaMV35S Term-LB

For both of the above plasmids, the expression cassettes in the firstT-DNA provide a means of selecting the transgenic donor lines afterAgrobacterium-mediated transformation. The second T-DNA provides thecomponents for crossing-mediated cassette exchange. Note that for bothconstructs, the inducible FLP expression cassette is outside the FRTsites and thus this is not transferred into the target site uponsuccessful exchange.

The recombination events having the transfer cassette are selected byvisual selection for vigorously growing, yellow-fluorescent calli,regenerated, grown to maturity and crossed to produce donor seed havingthe transfer cassette. Seed from a target event containing the T-DNAfragment from PHP20705 as well as seed from a donor event containing theT-DNA from Donor plasmid #1 above are planted and grown to maturity.Upon flowering, reciprocal crosses are made between the target and donorplants. The resultant seed are planted and screened for the newlyactivated phenotypes that indicate successful recombination at the twodissimilar FRT sites, in this case activation of bialaphos resistanceindicative of proper recombination at FRT5 and activation of Cyanfluorescence indicative of proper recombination at FRT87. Similarcrosses are done using target and transfer lines generated with PHP20807and Donor plasmid #2, respectively.

Example 8. Transformation of Bacterial Cells

The novel recombination sites provided herein can also be evaluated andused in bacterial cells, such as E. coli. Many commercially availablecompetent cell lines and bacterial plasmids are well known and readilyavailable. Isolated polynucleotides for transformation andtransformation of bacterial cells can be done by any method known in theart. For example, methods of E. coli and other bacterial celltransformation, plasmid preparation, and the use of phages are detailed,for example, in Current Protocols in Molecular Biology (F. M. Ausubel etal. (eds.) (1994) a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc.). For example, an efficientelectroporation protocol (Tung & Chow, Current Protocols in MolecularBiology, Supplement 32, Fall 1995) is summarized below.

Inoculate 100 ml LB medium with 1 ml E. coli overnight culture. Incubateat 37° C. with vigorous shaking until culture reached OD600=0.6. Chillculture on ice 30 min, pellet cells by centrifuging 4,000×g for 15 minat 4° C. Wash cell pellet twice with 50 ml ice-cold 10% glycerol. Afterfinal wash, resuspend cell pellet to a final volume of 0.2 ml inice-cold GYT medium (10% v/v glycerol; 0.125% w/v yeast extract; 0.25%w/v trytone). Electroporate in prechilled cuvettes using manufacturer'sconditions, for example 0.5 ng plasmid DNA/transformation using GenePulser (BioRad) set to 25 μF, 200 ohms, 2.5 kV. Immediately afterelectroporation, add 1 ml SOC medium and transfer cells to a culturetube. Incubate at 37° C. for 1 hr. Plate aliquots of cells on selectiveagar plates and incubate overnight at 37° C.

Example 9. Transformation of Yeast

The novel recombination sites provided herein can also be evaluated andused in yeast cells, from which FLP recombinase and FRT sites wereinitially isolated. Many commercially and/or publicly available strainsof S. cerevisiae are available, as are the plasmids used to transformthese cells. For example, strains are available from the American TypeCulture Collection (ATCC, Manassas, Va.) and includes the Yeast GeneticStock Center inventory, which moved to the ATCC in 1998. Other yeastlines, such as S. pombe and P. pastoris, and the like are alsoavailable. For example, methods of yeast transformation, plasmidpreparation, and the like are detailed, for example, in CurrentProtocols in Molecular Biology (F. M. Ausubel et al. (eds.) (1994) ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., see Unit 13 in particular). Transformation methods foryeast include spheroplast transformation, electroporation, and lithiumacetate methods. A versatile, high efficiency transformation method foryeast is described by Gietz & Woods ((2002) Methods Enzymol. 350:87-96)using lithium acetate, PEG 3500 and carrier DNA.

Example 10. Transformation of Mammalian Cells

The novel recombination sites provided herein can also be evaluated andused in mammalian cells, such as CHO, HeLa, BALB/c, fibroblasts, mouseembryonic stem cells and the like. Many commercially available competentcell lines and plasmids are well known and readily available, forexample from the ATCC (Manassas, Va.). Isolated polynucleotides fortransformation and transformation of mammalian cells can be done by anymethod known in the art. For example, methods of mammalian and othereukaryotic cell transformation, plasmid preparation, and the use ofviruses are detailed, for example, in Current Protocols in MolecularBiology (F. M. Ausubel et al. (eds.) (1994) a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc., see Unit9 in particular). For example, many methods are available, such ascalcium phosphate transfection, electoporation, DEAE-dextrantransfection, liposome-mediated transfection, microinjection as well asviral techniques.

Example 11. Methods for In Vitro Recombinational Cloning

In examples A, B, and C below, the two parental nucleic acid molecules(e.g., plasmids) are called the “insert donor” and the “vector donor.”The insert donor contains a segment that will become joined to a newsequence contributed by the vector donor. The recombination eventproduces two daughter molecules: the first referred to as the product(the desired new clone) and the second referred to as the by-product.

In the examples below, two pairs of plasmids are constructed to performthe in vitro recombinational cloning method in two different ways. Onepair of plasmids, Plasmid A and plasmid B, are constructed with a FRTsite and a lox site, to be used with Cre and FLP recombinase. The otherpair of plasmids, Plasmid D and Plasmid E, are constructed to containthe FRT (wild type) site for FLP, and a second mutant FRT site (SEQ IDNO:40), which differs from the FRT wild type site in 3 out of 30 basestotal. In this example, each plasmid comprises a set of functionalrecombination sites wherein the recombination sites are dissimilar andnon-recombinogenic with respect to one another.

Buffers:

Various known buffers can be used in the reactions. For restrictionenzymes, it is advisable to use the buffers recommended by themanufacturer. Alternative buffers can be readily found in the literatureor can be devised by those of ordinary skill in the art. One exemplarybuffer for lambda integrase comprises 50 mM Tris-HCl, at pH 7.5-7.8, 70mM KCl, 5 mM spermidine, 0.5 mM EDTA, and 0.25 mg/ml bovine serumalbumin, and optionally, 10% glycerol. An exemplary buffer for P1 Crerecombinase comprises 50 mM Tris-HCl at pH 7.5, 33 mM NaCl, 5 mMspermidine, and 0.5 mg/ml bovine serum albumin and an exemplary bufferfor FLP is discussed above in Example 2. An exemplary buffer for Cre andFLP recombinases comprises 50 mM Tris-HCL at pH 7.5, 70 mM NaCl, 2 mMMgCl₂, and 0.1 mg/ml BSA, (Buchholz et al. (1996) Nucleic Acids Res.24:4256-4262). The buffer for other site-specific recombinases areeither known in the art or can be determined empirically by the skilledartisans, particularly in light of the above-described buffers.

A. Recombinational Cloning Using FLP Recombinase

Two plasmids are constructed. The donor plasmid (plasmid A) comprises inthe following order: a wild type FRT site, a constitutive drug marker(chloramphenicol resistance), an origin of replication, a constitutivelyexpressed gene for the tet repressor protein (tetR), a FRT 5 site, and aconditional drug marker (kanamycin resistance whose expression iscontrolled by the operator/promoter of the tetracycline resistanceoperon of transposon Tn10). E. coli cells containing plasmid A areresistant to chloramphenicol at 30 μg/ml, but sensitive to kanamycin at100 μg/ml.

The insert donor plasmid (plasmid B) comprises in the following order:the wild type FRT site, a different drug marker (ampicillin resistance),the FRT 5 site, an origin, and a multiple cloning site.

About 75 ng each of plasmid A and B are mixed in a total volume of 30 μlof FLP reaction buffer. Two 10 μl aliquots are transferred to new tubes.One tube receives FLP protein. Both tubes are incubated at 37° C. for 30minutes, then 70° C. for 10 minutes. Aliquots of each reaction arediluted and transformed into DH5α. Following expression, aliquots areplated on 30 μg/ml chloramphenicol; 100 μg/ml ampicillin plus 200 μg/mlmethicillin; or 100 μg/ml kanamycin.

Colonies that are chloramphenicol resistant, ampicillin resistant, andkanamycin sensitive under went the recombination reaction and comprisethe newly generated product vector (plasmid C). Plasmid C comprises inthe following order: the wild type FRT site, the constitutive drugmarker (chloramphenicol resistance), the origin of replication, theconstitutively expressed gene for the tet repressor protein (tetR), theFRT 5 site, and the ampicillin resistance marker.

To confirm the structure of the product vector (plasmid C), coloniesthat are chloramphenicol resistant, ampicillin resistant, and kanamycinsensitive are picked and inoculated into medium containing 100 μg/mlkanamycin. Minipreps are performed and the miniprep DNAs are cut withthe appropriate restriction enzymes and electrophoresed. Plasmid C canbe identified by based on the size predicted for the Product plasmid andthe resulting fragments of the restriction enzyme digest.

B. Recombinational Cloning Using FLP Recombinase and Cre Recombinase

The plasmids of this method are analogous to those above, except thatPlasmid D, the vector donor plasmid, contains a loxP site in place ofthe wild type FRT site, and Plasmid E, the insert donor plasmid,contains the loxP site in place of the wild type FRT site.

About 500 ng of Plasmid E and Plasmid D are ethanol precipitated andresuspended in 40 μl buffer Cre/FLP reaction buffer (described above).Reactions are incubated at 37′ C for 30 minutes and then at 70′ C for 10minutes. TE buffer (90 μl; TE: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) isadded to each reaction, and 1 μl each is transformed into E. coli DH5α.The transformation mixtures are plated on 100 μg/ml ampicillin plus 200μg/ml methicillin; 30 μg/ml chloramphenicol; or 100 μg/ml kanamycin.

Colonies that are chloramphenicol resistant, ampicillin resistant, andkanamycin sensitive under went the recombination reaction and comprisethe newly generated product vector (plasmid F). Plasmid F comprises inthe following order: the wild type loxP site, the constitutive drugmarker (chloramphenicol resistance), the origin of replication, theconstitutively expressed gene for the tet repressor protein (tetR), theFRT 5 site, and the ampicillin resistance marker.

To confirm the structure of the product vector (plasmid F), coloniesthat are chloramphenicol resistant, ampicillin resistant, and kanamycinsensitive are picked and inoculated into medium containing 100 μg/mlkanamycin. Minipreps are performed and the miniprep DNAs are cut withthe appropriate restriction enzymes and electrophoresed. Plasmid F canbe identified by based on the size predicted for the Product plasmid andthe resulting fragments of the restriction enzyme digest.

C. In Vitro Recombinational Cloning to Subclone the ChloramphenicolAcetyl Transferase Gene into a Vector for Expression in Eukaryotic Cells

An insert donor plasmid, Plasmid G, is constructed, comprising in thefollowing order: a wild type FRT site, a chloramphenicol acetyltransferase gene of E. coli lacking a promoter, the FRT 5 site, anorigin of replication, and a constitutive drug marker (ampicillinresistance).

A vector donor plasmid, Plasmid H, is constructed, which comprises inthe following order: kanamycin resistance gene, origin of replication,the cytomegalovirus eukaryotic promoter, a wild type FRT site, theconstitutively expressed gene for the tet repressor protein (tetR), achloramphenicol resistance gene, and the FRT 5 site. One microliteraliquots of each plasmid, typically about 50 ng crude miniprep DNA, arecombined in a 10 μl reaction containing a FLP reaction buffer and FLPrecombinase. After incubation at 30° C. for 30 minutes and 75° C. for 10minutes, one microliter is transformed into competent E. coli strainDH5α (Life Technologies, Inc.). Aliquots of transformations are spreadon agar plates containing 200 μg/ml kanamycin and incubated at 37° C.overnight. An otherwise identical control reaction contains the vectordonor plasmid only.

To confirm the structure of the product vector (plasmid I), miniprepsare performed and the miniprep DNAs are cut with the appropriaterestriction enzymes and electrophoresed. Plasmid I can be identified bybased on the size predicted for the product plasmid and the resultingfragments of the restriction enzyme digest to confirm thechloramphenicol acetyl transferase is cloned downstream of the CMVpromoter.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more than one element.

That which is claimed:
 1. An isolated library comprising a population of plasmids wherein each plasmid in the population comprises a first common selectable marker; and each plasmid in the population comprises a member of a population of modified FRT recombination sites, wherein each member of the population of modified FRT recombination sites comprises a spacer region comprising at least one nucleotide alteration in SEQ ID NO:43, wherein the population of plasmids comprises at least about 100 distinct members of the plasmid population.
 2. The isolated library of claim 1, wherein the modified FRT recombination sites of the population are recombinogenic.
 3. The isolated library of claim 1, wherein the modified FRT recombination sites do not include modified FRT recombination sites with a spacer region selected from the group consisting of SEQ ID NOS: 4, 44, 55, 56, 57, 58, 59, 60, 61, 62, 63, and
 64. 4. The isolated library of claim 1, wherein the population of plasmids comprises at least one modified recombinogenic FRT site selected from the group consisting of: a) a polynucleotide comprising a spacer region selected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, and: b) a modified recombinogenic FRT site selected from the group consisting of SEQ ID NO:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and
 38. 5. A kit comprising the population of plasmids of claim 1, further comprising a second population of plasmids wherein each plasmid in the second population comprises a second common selectable marker, wherein the first and the second selectable markers are distinct; and, each plasmid in the second population comprises a member of the population of modified FRT recombination sites.
 6. The kit of claim 5, wherein the kit further comprises a FLP recombinase or a polynucleotide encoding the FLP recombinase.
 7. The kit of claim 5, wherein at least one member of the first population, the second population, or both populations of modified FRT recombination sites comprises: a) a spacer region selected from the group consisting of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18; and, b) a modified recombinogenic FRT site selected from the group consisting of SEQ ID NOS:21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and
 38. 8. The kit of claim 5 wherein each member of the first and the second population of modified FRT recombination site is recombinogenic.
 9. A method for generating a library of molecules comprising a) providing a population of modified FRT recombination sites, wherein each member of the population of modified FRT recombination sites comprises a spacer region comprising at least one nucleotide alteration in SEQ ID NO:43; and, b) contacting the population of modified FRT recombination sites with a population of plasmids having a common selectable marker under conditions for the insertion of the population of modified FRT recombination sites into the population of plasmids, such that each of the plasmids of the population comprises a single member of the population of modified FRT recombination sites to generate a library of molecules.
 10. A method to select a recombinogenic modified FRT recombination site comprising: a) providing a first population of plasmids wherein each plasmid in the first population comprises a common first selectable marker; and, each plasmid in the first population comprises a member of a population of modified FRT recombination sites, wherein each member of the population of randomized modified FRT recombination sites comprises a spacer region comprising at least one nucleotide alteration in SEQ ID NO:43; b) providing a second population of plasmids wherein each plasmid in the second population comprises a common second selectable marker, wherein the first and the second selectable markers are distinct; and, each plasmid in the second population comprises a member of the population of modified FRT recombination sites; c) combining the first population of plasmids with the second population of plasmids in the presence of a FLP recombinase under conditions where site-specific recombination can occur; and, d) selecting for a co-integrant plasmid comprising the first and the second selectable marker, the co-integrate plasmid comprising the modified FRT recombination site.
 11. The method of claim 10, wherein the co-integrant plasmid comprises a functional modified FRT recombination site.
 12. The method of claim 10, further comprising isolating the co-integrant plasmid.
 13. The method of claim 10, further comprising characterizing the modified FRT recombination site of the co-integrant plasmid.
 14. The method of claim 13, wherein characterizing the modified FRT recombination site comprises a method selected from the group consisting of determining excision efficiency, determining recombination specificity, and sequencing the modified FRT recombination site.
 15. The method of claim 9, wherein the first population of plasmids and the second population of plasmids are combined in an equimolar ratio. 